CN1238013A

CN1238013A - Method for detection of metastatic prostate cancer

Info

Publication number: CN1238013A
Application number: CN 97199740
Authority: CN
Inventors: D·J·廷达尔; C·Y·F·尤恩格; D·J·麦克科米克; G·G·克利; M·S·萨狄; A·库马尔; H·G·里坦豪斯; R·L·沃尔福特
Original assignee: Hybritech Inc; Mayo Foundation for Medical Education and Research
Current assignee: Hybritech Inc; Mayo Foundation for Medical Education and Research
Priority date: 1996-11-14
Filing date: 1997-11-14
Publication date: 1999-12-08
Also published as: JP2001503991A; WO1998021365A3; CA2271770A1; WO1998021365A2; EP0941368A2; AU5260298A; AU737659B2

Abstract

Prostate cancer is detected by measuring the presence of prostate-specific glandular kallikrein hK2 polypeptide hK2 RNA in a physiological sample.

Description

The detection method of metastatic prostate cancer

The government rights statement

The present invention is (fund CA70893 and the DK41995) that finishes under the subsidy that United States Government is undertaken by National Institutes of Health.Government may enjoy some right in the present invention.

Background of invention

Kallikrein plays a role in specific polypeptide precursor translation post-treatment becomes the process of its biologically active form, and the gland kallikrein is its subgroup.In the mankind, determined three members of this family, their some characteristic also obtains identifying (Clements, internal secretion comment, 10,343 (1989); Clements, molecular cell incretology, 99,1 (1994); Jones etc., internal secretion journal, 127,481 (1992).HKLK1 genes encoding tissue kallikrein protein matter hK1, the distinctive gland kallikrein protein of hKLK2 genes encoding prostate gland matter hK2, the distinctive antigen protein hK3 of hKLK3 genes encoding prostate gland (PSA).MRNA Northern hybridization analysis shows that hK2 and PSA mainly express in human prostate, and hK1 is at pancreas, and glandula submandibularis is expressed (Chapdelaine etc., FEBS communication, 236,205 (1988) in kidney and other non-prostata tissue; Young etc., biological chemistry, 31,818 (1992)).

Nucleotide sequence homology between HKLK2 and the hKLK3 exon is 80%, and the homology of the nucleic acid glycosides sequence between hKLK2 and the hKLK1 exon is 65%.The amino acid sequence homology that hK2 and PSA infer is 78%, and the amino acid sequence homology that hK2 and hK1 infer is 57%.In addition, the aminoacid sequence prompting hK2 that hK2 infers may be a kind of trypsin-like proteolytic enzyme, and PSA is a kind of chymotrypsin-like proteolytic enzyme.

The PSA level is widely used as the prediction index of prostate cancer.Yet,, detect the PSA level that raises and can not distinguish this two kinds of diseases because blood-serum P SA concentration all raises in prostate cancer (pCa) patient or benign prostatic hyperplasia (BPH) patient.In addition, the high homology of hK2 and PSA has proposed query to the specificity of the antibody that is used to detect the PSA level at present.If hK2 level and pCa in the circulation and BPH are irrelevant, the PSA preparation of hK2 is arranged by pollution so, or by with the antibody of hK2 homologous PSA zone generation, can cause false positive results.

Though extensively think at present, it is to determine the most effective means of prostate cancer significant clinically and that the organ scope limits that blood-serum P SA detects with combining of finger examination per rectum (DRE), and PSA, DRE and prostate gland ultrasound investigation can only be found the part prostate cancer.For example, the patients with prostate cancer of the underwent operative of as many as 40% treatment is found to be clinical recessiveness subsequently.In addition, the sense of organization oncogenesis rate of determining on the postmortem basis is than the incidence of prostate cancer is higher significantly clinically.Moreover about 30% it is believed that it is among the patient of localized prostate cancer the secret metastasis of (at a distance) (Moreno etc., cancer research, 52,6110 (1992)) to be arranged.In these patients, 80% patient is in the recurrence of having experienced after the treatment on the biochemical meaning, and promptly the PSA level raises, local recurrence or the obviously appearance or the morbidity of general symptom.

Concerning the patient who knows transfer, operative treatment is not a kind of suitable therapeutic modality.The screening method that uses in order to assess early stage transfer often can not find to have among the patient a big subgroup of invading capsula prostatica and this local invasion and attack pathology of seminal vesicle.When not detecting obvious metastasis as yet with ordinary method, the prostate tumor cells in that immunohistochemical method can be used to identify micrometastasis or the circulation.But immunohistochemical method is loaded down with trivial details, for the required susceptibility of early detection shortage of transfer or local infection prostate cancer.

Like this, need a kind of energy early detection have the method for the prostate cancer cell of metastatic potential.In addition, need a kind of method that can determine the prostate cancer emergence period before to patient treatment, need especially a kind ofly can independently play a role and can combine the prostate cancer sign of using with PSA.

Summary of the invention

The invention provides a kind of diagnostic method that is used for detecting hK2 DNA, wherein the existence of prostate cancer cell can take place related with the detection of hK2 RNA in this sample in the physiologically sample.Because it is peculiar that the expression of hK2 is a prostata tissue, so theoretically, when local invasion and attack or metastasis do not take place, or when all prostata tissues (optimum and pernicious) all have been eliminated or have destroyed, in the cell of body fluid or should not detect the expression of hK2 in the non-prostata tissue.Present method comprises, RNA by human physiologically sample source produces a certain amount of DNA through reverse transcription (RT), the DNA that obtains is contacted with numerous Oligonucleolide primers, preferred at least two kinds of Oligonucleolide primers, wherein at least a is the special oligonucleotide of hK2, produces a certain amount of amplification hK2 DNA by an amplified reaction.Preferred amplified reaction is polymerase chain reaction (PCR).Detect the hK2 DNA that whether has amplification subsequently.As mentioned below, through after the RT-PCR amplification, in the hemocyte existence of hK2 DNA cloning product relevant with prostate cancer, that is to say 67% patients with prostate cancer expression hK2,17% patient expresses PSA, 17% patient expresses hK2 and PSA simultaneously.Preferably, the sample source that is used to detect is in human tissue, as prostate gland, and capsula prostatica, seminal vesicle, marrow or lymphoglandula, the another kind of sample source that preferably is used to detect contains cells physiological liquid in the mankind, as blood, serum or seminal fluid.

" the hK2 DNA of amplification " used herein is meant through the hK2 DNA in the sample behind the amplified reaction, and its quantity is Duoed 10 times, preferred 10 than the amount of hK2 DNA in the preceding same sample of amplification ⁴Doubly, more preferably 10 ⁶Doubly.

" primer that oligonucleotide that hK2 is special or hK2 are special " used herein refers to the section of DNA sequence, be different among this sequence and the SEQ ID NO:4 between the zone of nucleotide sequence (No. 23 sequences) of the hK3 that encodes and have about at least 80% sequence identity or homology, preferably about at least 90%, more preferably about at least 95%.Oligonucleotide of the present invention or primer have the Nucleotide about 7-50 at least, the Nucleotide about preferred 10-40 at least, the more preferably Nucleotide about 15-35 at least.Preferably, 3 ' end of Oligonucleolide primers of the present invention has 7 Nucleotide and SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 existence about at least 80% at least, and more preferably about at least 85%, also will be preferably the about at least 90% identical.Oligonucleotide of the present invention may also contain and the irrelevant sequence of hK2 nucleotide sequence, as the sequence of the restriction enzyme enzyme recognition site of may encoding.Special oligonucleotide of preferred hK2 of the present invention comprises SEQ IDNO:14, and the special oligonucleotide of another preferred hK2 of the present invention comprises SEQ ID NO:17, and the special oligonucleotide sequence of another preferred hK2 of the present invention comprises SEQ ID NO:18.

A kind of preferred diagnostic method of the present invention is the RT-PCR detection and the RT-PCR detection of other relating to prostate cancers because of the product transcript in conjunction with the hK2 transcript.The joint-detection of two or more gene products makes diagnosis more accurate, and more staging information can be provided.Joint-detection also helps distinguishing the cell with invasive growth potentiality and has more the inert cell.A particularly preferred embodiment of the inventive method is the RT-PCR of hK2 RNA to be detected RT-PCR with PSA RNA detect and combine.

The present invention further provides the diagnostic method of a kind of hK2 of detection RNA.This method comprises from the physiologically sample that derives from the people extracts RNA, the RNA reverse transcription that extracts is become DNA, with this DNA with a certain amount of at least two kinds effectively the oligonucleotide of DNA amplification combine, to produce a certain amount of amplification hK2 DNA, wherein having a kind of oligonucleotide at least is the special oligonucleotide of hK2.Detect the hK2 DNA that whether has amplification subsequently.The existence indication this person of the hK2 DNA of amplification suffers from metastatic prostate cancer.

The existence of hK2 RNA in body fluid or the non-prostata tissue, or the level of hK2 RNA raises the transitivity pathology that may reasonably indicate existence not diagnosed out in the past in time.The early detection of transitivity pathology provides lead time for considering alternative treatment plan, may also not exist in surgical stages comprising those, and the treatment plan that is developed afterwards.Like this, the invention provides a kind of method of monitoring the prostate cancer development.

This method comprises, the RNA reverse transcription that derives from the physiologically sample of patients with prostate cancer is become DNA, a certain amount of this DNA is combined with at least two kinds of oligonucleotide (the special oligonucleotide of the wherein at least a hK2 of being) a certain amount of, this DNA that can effectively increase, to produce a certain amount of amplification hK2 DNA, the hK2 DNA of qualitative detection or quantitative assay amplification.At least afterwards point is sometime got another duplicate samples, and the hK2 DNA that increases is carried out qualitative or detection by quantitative.Then, the amplification amount to the hK2 DNA that obtains two different time points at least compares.

And provide the method for the human prostate cancer being carried out pathological staging.This method comprises, the RNA reverse transcription that derives from the physiologically sample of patients with prostate cancer is become DNA, a certain amount of this DNA is combined with at least two kinds of oligonucleotide (the special oligonucleotide of the wherein at least a hK2 of being) a certain amount of, this DNA that can effectively increase, to produce a certain amount of amplification hK2 DNA, then qualitative detection or quantitative assay the amplification hK2 DNA.The index that the existence of amplification hK2 DNA or quantity are the prostate cancer pathological stage.

Another embodiment of the invention is to provide a kind of monitoring scheme for the therapeutic intervention that comprises hormonotherapy.For example, because the expression of hK2 is an androgen-dependent, so the level of hK2 DNA in peripheral blood, other bodily tissue or body fluid can be used as the index of intermittent male sex hormone treatment or male sex hormone stimulation test, wherein make the patient in the male sex hormone stimulation test, temporarily be in a kind of male sex hormone and cross M-ARY, stimulating any prostate cancer cell of remaining to produce the hK2 of certain level, thereby detect the existence of these cells.Referring to T.K.Takayama etc., oncology discussion, 21,542-553 (1994) and the document of quoting thereof.Preferably, in the hormonotherapy process, periodically monitor the hK2 level.Before the treatment beginning or after the treatment end hK2 level being carried out periodicity measures and may be highly profitable.

And providing a kind of has the physiologically sample of hK2 RNA to carry out the diagnostic kit that hK2 RNA detects to suspection.This test kit comprises and contains following packing: (a) first of known quantity kind of special oligonucleotide of hK2, this oligonucleotide comprise the Nucleotide about 7-50 at least, and this oligonucleotide has at least 80% left and right sides identical with SEQ ID NO:4; (b) second of known quantity kind of special oligonucleotide of hK2, this oligonucleotide comprise the Nucleotide about 7-50 at least, and this oligonucleotide has at least 80% left and right sides identical with the nucleotide sequence that is complementary to SEQ ID NO:4.

The present invention further provides comprise SEQ ID NO:22 a kind of through separate, the peptide of purifying, its bioactive variant that has that bioactive subunit or it are arranged.The present invention further provides comprise SEQ ID NO:26 a kind of through separate, the peptide of purifying, its bioactive variant that has that bioactive subunit or it are arranged.Also provide can with the protein that comprises the above-mentioned peptide of the present invention or polypeptide specific reaction a kind of through separate, antibody purified or antibody preparation.

" bioactive subunit is arranged " speech of peptide of the present invention used herein, preferably finger has the subunit of the peptide of SEQ ID NO:22, this subunit has active about 10% the activity of the peptide of tool SEQ ID NO:22 at least, preferred about at least 50% activity, more preferably about at least 90% activity.The active mensuration of peptide of the present invention can adopt the method for understanding thoroughly in this area, and it causes the ability of sequence-specific immune response when this peptide is in being administered to as organisms such as goat, rabbit, sheep or mouse.

Peptide of the present invention used herein " has a bioactive variant " speech preferably refers to have the identical or homology in 80% left and right sides at least with SEQ IDNO:22, preferably has the identical or homology in 90% left and right sides at least, more preferably has the identical or homologous peptide in 95% left and right sides at least.The bioactive variant that has of peptide of the present invention has 10% left and right sides biological activity of the peptide of tool SEQ ID NO:22 at least, preferably has about 50% activity at least, more preferably has about 90% activity at least.The activity of peptide variant of the present invention can be measured by aforesaid method.

The present invention further provides a kind of method that detects or measure metastatic prostate cancer in the human non-prostata tissue sample.This method comprises the cytomixis in a certain amount of reagent and the mammalian tissues sample is formed a kind of divalence mixture that comprises reagent and cell that this reagent only combines and do not combine with hK3 with the hK2 polypeptide.The mixture that forms in the sample is carried out qualitative detection and quantitative assay.The existence of this mixture or quantity are the indications that the micrometastasis prostate cancer exists." micrometastasis " used herein is meant local aggressive pathology, generally includes the invasion and attack of capsula prostatica or seminal vesicle, or recessive pathology.A kind of preferred reagent is antibody in this method." antibody " speech comprises the monoclonal antibody of humans and animals, and polyclonal antibody preparation, and antibody fragment and synthetic antibody comprise recombinant antibodies and chimeric antibody, and it comprises humanized antibody again, anti-allotypic antibody and derivative thereof.For preparation only with hK2 not with hK3 bonded antibody, can be with isolating hK2 polypeptide, isolating hK2 peptide and their variant or subunit prepare antibody population.These antibody are used as and detect and quantitative assay derives from tissue sample such as marrow, lymphoglandula and such as the basis that derives from the direct or competitive assay of hK2 polypeptide (or protein) in the cell sample that contains cells physiological liquid subsequently.

The accompanying drawing summary

Accompanying drawing 1 has been described the aminoacid sequence of sophisticated wild-type hK2 (SEQ ID NO:1) and hK3 (SEQ ID NO:7).

Accompanying drawing 2 has been described wild-type pphK2 (SEQ ID NO:3 and SEQ ID NO:4), the aminoacid sequence of phK2 (SEQ ID NO:5 and SEQ ID NO:6) and hK2 (SEQ ID NO:1 and SEQ ID NO:2) and corresponding nucleotide sequence.No. 217 codons (GCT, L-Ala) mark with runic and underscore.

Accompanying drawing 3 is pGT expression vectors--pGThK2 and pGThK2 ^V217Sketch.

Accompanying drawing 4 is phK2 ^V217The color atlas of purifying.(A) transfection has coding pphK2 ^V217The DEAE color atlas of 7 days consumption substratum of the AV12 cell cultures of carrier.With sample on the consumption media samples is in the bicarbonate buffer of pH8, adopt the salt gradient wash-out.Represent A with solid line ₂₈₀Elution curve, dotted line is represented the ELISA result of each post elutriated fraction part, promptly with the anti-pphK2 antibody of rabbit drying each post fraction on titer plate is developed the color.(B) compile the hydrophobic interaction curve of DEAE fraction.The 24-30 fraction of compiling DEAE chromatography eluate in (A) concentrates, in the hydrophobic interaction post (HIC) of last sample in the 1.2M sodium sulfate, and with the salt concn gradient elution of reduction gradually.Solid line is represented elution curve (A ₂₈₀), dotted line is represented the ELISA test result of each elutriated fraction part, with the anti-hK2 antibody of rabbit drying each fraction on titer plate is developed the color.(C) sample is gone up in pharmacia S12 size-exclusion column in the concentrated back of the fraction that contains hK2 at 22 minutes peaks in (B), collect elutriated fraction and carry out SDS-polyacrylamide gel electrophoresis (SDS/PAGE) analysis, wherein 19.4 minutes elution peak shows as the SDS/PAGE homogeneous.

The hK2 of accompanying drawing 5 expression purifying and the SDS/PAGE of PSA analyze.To add (R) or add 1.5 milligrams of (N) the 1% beta-mercaptoethanol phK2 of purifying ^V217Or PSA boils, and carries out SDS/PAGE then on the gel of 4-20%, makes the protein band colour developing with argentation.

Accompanying drawing 6 has been described phK2 ^V217Concanavalin A matter A coloration result.The desired location of arrow indication phK2.Use ZCE (monoclonal antibody of a kind of anti-CEA) and bovine serum albumin (BSA) example as glycosylation and non-glycosylated protein respectively, it is glycosylated protein that the phK2 swimming lane this protein of bar carrying means occurred in desired location.

Accompanying drawing 7 expressions make hK2 by the trypsinase cutting ^V217Precursor protein matter is converted into mature protein.At 37 ℃, under the 100mM borate buffer solution condition of pH8, with trypsin 1%w/w) and phK2 ^V217 Common incubation 10 minutes carries out HIC/HPLC then and detects.Broken broken line is represented the phK2 before the trypsinase incubation ^V217Curve, solid line are represented the graph curve of phK2 behind the trypsinase incubation.Two curves are superposeed to compare.Determine the homogeny of two kinds of protein forms with the protein N terminal sequencing.

Accompanying drawing 8 has been described the Western results of hybridization to seminal fluid with monoclonal antibody (mAb) hK1G586.1.The seminal fluid of handling is diluted among the PBS at 1: 1 centrifugal 20 minutes of 10000xg.Supernatant carries out SDS/PAGE on the gel of 8-25%, adopt the Phast System electrophoresis system of Pharmacia.Protein transduction is moved on on the nitrocellulose filter, and through the common incubation of the HK1G586.1 of G-protein purification antibody (1 μ g/ml), add sheep anti-mouse igg-HRT (1: 1000) subsequently.Trace develops the color with the ECL detection system of Amersham.

Accompanying drawing 9 has been represented the time curve that hK2 expresses in the AV12 cell.No. 27 clones are cultured to about 60-70% degree of covering with the AV12-hK2 cell, wash cell with HBSS, add the HH4 substratum of serum-free.Reclaim to consume substratum every day, be splined on after concentrating on 12% the glue and carry out SDS/PAGE.Behind the protein electrotransfer, detect with monoclonal antibody HK1D106.4 or HK1G464.3.HK1D106.4 can detect phK2 and hK2 (1: 1000), and HK1G464.3 can detect phK2 (1: 1000).Add sheep anti-mouse antibody IgG-HRP (1: 500) subsequently.Press operational manual explanation, trace is developed the color with the ECL system of Amersham company.PhK2 with purifying ^V217And hK2 ^V217Compare, arrow indication place is the position of hK2.

The time curve of hK2 variant is expressed in accompanying drawing 10 expressions in the AV12 of transfection cell.Grow to the AV12-hK2 that about 60-70% merges ^V217After cell washes with HBSS, add the HH4 substratum of serum-free.Every day results consume substratum, are splined on after concentrating on 12% the glue to carry out SDS/PAGE.Behind the protein electrotransfer, detect with monoclonal antibody HK1D106.4 and HK1G464.3.Anti-with sheep anti-mouse igg-HRP (1: 500) as two, press operational manual, with ECL (Amersham) trace is developed the color.PhK2 with purifying ^V217And hK2 ^V217Compare.The position of arrow indication hK2.

Accompanying drawing 11 is time curves of hK2 expression and cell viability.No. 27 clones of AV12-hK2 cell grow to 60-70% and merge, and with the HBSS flushing, add the HH4 substratum of serum-free.Every day, results consumed substratum, and anti-as one with HK1D106.4 or HK1G464.3, sheep anti-mouse igg-HRP is anti-as two, through the concentration of ELISA reaction assay hK2.(Sigma.St.Louis MO) carries out color reaction with OPD.Count survivaling cell every day with trypan blue exclusion.

Accompanying drawing 12 has been described the expression of hK2 in PC3 and the DU145 cell.Transfection has the PC3 of pGThK2 and DU145 cell to grow to about 60-70% fusion, is resuspended in after the washing in the HH4 substratum of serum-free.Collect the consumption substratum that transfection has the DU145 cell of pGThK2 after resuspended 3 days, collect the consumption substratum that transfection has the PC3 cell of pGThK2 after resuspended 5 days.Consume to be splined on after substratum concentrates on 12% the glue and carry out the SDS/PAGE electrophoresis.As above-mentioned method, behind the protein electrotransfer, detect as probe with HK1D106.4 and HK1G464.3.PhK2 with purifying ^V217And hK2 ^V217Compare.The position of arrow indication hK2.

Figure 13 has described the expression of hK2 in through the AV12 cell clone of selecting that contains hK2.The 10th, 27,31 and No. 32 clones that contain the AV12 cell of hK2 grow to about 60-70% and merge, wash with HBSS, the HH4 substratum that adds serum-free is then cultivated after 7 days to collect and is consumed substratum, concentrates and be splined on 12% the gel to carry out the SDS/PAGE electrophoresis.The protein electrotransfer, and detect as probe with HK1D106.4 or HK1G464.3.Anti-with sheep anti-mouse igg-HRP (1: 500) as two, trace is developed the color with ECL (Amersham) according to operation instructions.PhK2 with purifying ^V217And hK2 ^V217In contrast, the position of arrow indication hK2.

Figure 14 has described at the AV12-hK2 through screening ^V217PhK2 among the clone ^V217Expression.AV12 the 2nd, 3, and 4,45 and No. 48 clones' cell grows to about 60-70% and merges, and with the HBSS flushing, adds the HH4 substratum of serum-free.Add serum free medium and collects after 7 days and consume substratum, be splined on after concentrated in 12% the gel and carry out the SDS/PAGE electrophoresis.The protein electrotransfer is detected as probe with HK1D106.4 or HK1G464.3.Anti-with sheep anti-mouse igg-HRP (1: 500) as two, according to operation instructions, trace is developed the color with ECL (Amersham).PhK2 with purifying ^V217And hK2 ^V217Compare.The position of arrow indication hK2.

Figure 15 has described hK2 ^V217, hK2 and PSA are to the amide hydrolysis specificity of 210-236 position residue among the hK2.0.63mM synthetic peptide is following to 1 μ g/ml hK2,40 μ g/ml hK2 at 37 ℃ ^V217Or 100 μ g/ml PSA digestion spend the night, with RP-HPLC separating digesting product, with each peak stdn with the properties of cutting relatively.

Accompanying drawing 16 has been described hK2 and the specificity of PSA to different peptide substrates.The peptide bond that hollow arrow indication is cut by PSA, filled arrows are represented the peptide bond that cut by hK2.No. 1 peptide is represented 210-236 amino acids residue among the hK2, No. 2 peptide is represented proangiotensin 1-14 amino acids residue (being feritin substrate decapeptide), No. 3 peptides are represented among the phK2-7-+7 amino acids residue, No. 4 peptide is represented 41-56 amino acids residue among the hK2, No. 5 peptides are represented the aminoacid sequence of oxidized form Regular Insulin β chain, and No. 6 peptide is represented 196-213 amino acids residue among the PMSA.

Accompanying drawing 17 has been described phK2 ^V217Can be activated by hK2, but can not be by hK2 ^V217Activate.PhK2 ^V217Contain the leader peptide sequences VPLIQSR of precursor protein matter, this sequence is at hK2 ^V217In do not exist.A represents the phK2 with the common incubation of 1% (weight ratio) hK2 ^V217B is contrast, wherein phK2 ^V217With 40% (weight ratio) hK2 ^V217 Common incubation 6 hours.

Accompanying drawing 18 has been described the Western engram analysis with the hK2 of the common incubation of proteinase inhibitor.Each sample is separated in the gradient SDS/PAGE of 8-25%, change film and detect with HK1G586.1.Under 37 ℃, with hK2 and the common incubation of following inhibitor 4 hours: 1 swimming lane, chymotrypsin inhibitor (ACT); 2 swimming lanes, α 2 antiplasmins; 3 swimming lanes, antithrombin; 4 swimming lanes, α 1 proteinase inhibitor (antitrypsin); 5 swimming lanes, alpha2 Macroglobulin; 1 swimming lane and 2 swimming lanes have shown infers that molecular weight is a kind of covalent complex of 90-100KD.The concentration of serpin is 20 μ M, and macroglobulin is 2.8 μ M, and hK2 is 0.175 μ M.5 swimming lanes have shown the mixture of larger molecular weight, and what their were represented is the covalent complex of hK2 and alpha2 Macroglobulin.

Accompanying drawing 19 has been described hK2 forms mixture in human serum situation.Western trace and the common incubation of human serum with hK2 and PSA.Survey the hK2 sample with HK1G586.1, survey the PSA sample with the anti-PSA monoclonal antibody of PSM773.1 swimming lane to 6 swimming lane contains the hK2 sample, and 7 swimming lanes and 8 swimming lanes are the PSA sample.1 swimming lane is represented the hK2 contrast, 2 swimming lanes are and 4 hours hK2 of the common incubation of ACT, 3 swimming lanes are not for adding the serum contrast of proteolytic enzyme, 4 swimming lanes contain and 15 minutes hK2 of the common incubation of serum, 5 swimming lanes are and 4 hours hK2 of the common incubation of serum, 6 swimming lanes are and 4 hours hK2 of the common incubation of the alpha2 Macroglobulin of purifying that 7 swimming lanes are and 4 hours PSA of the common incubation of serum that 8 swimming lanes contain 4 hours the PSA of the common incubation of α-2 macroglobulin with purifying.

Figure 20 has described the immunoreactivity (n=257) of monoclonal antibody HK1G586 in untreated human prostate.

Figure 21 has described monoclonal antibody HK1G586 in the immunoreactivity (n=7) in the human prostate of the hungry treatment of male sex hormone.

Figure 22 has described (A) from the RNA of the LNCaP cell extraction that is diluted in whole blood, the mRNA of PSA and hK2 is carried out RT-PCR detect, (B) among the RNA that extracts from following patient's complete blood cell, the mRNA of PSA and hK2 is carried out RT-PCR detect: No. 17 patients, 31 years old, male sex's contrast; No. 21 patients, 58 years old, clinical stage B; No. 24 patients, 83 years old, known had a metastasis (D2); No. 26 patients, 75 years old, pathology stage C (+edge); No. 28 patients, 57 years old, pathology stage C (+edge); No. 49 patients, 64 years old, pathology stage C (+seminal vesicle); No. 59 patients, 31 years old, male sex's contrast; No. 60 patients, 73 years old, known had a metastasis.

Detailed Description Of The Invention

HK2 and the PSA high homology on aminoacid sequence, and the expression of hK2 and PSA is confined to the fact of prostatic cell substantially, prompting detect hK2 and PSA in tissue sample existence or measure its content, or the level of detection hK2 transcript in including cells physiological liquid (as blood) or tissue sample (as lymphoglandula), help diagnosis and monitoring prostate cancer (pCa).Definition

" hK2 polypeptide " speech used herein comprises the preceding hK2 propolypeptide of reorganization, preceding hK2 polypeptide and sophisticated hK2 polypeptide, sophisticated hK2 polypeptide comprises aminoacid sequence among Fig. 1 (SEQ ID NO:1), and with SEQ ID NO:1 in basic " variant " polypeptide that at least 90% homology is arranged with the zone (being the zone that is not marked by lines among Fig. 1) that comes from hK3.Variant hK2 polypeptide of the present invention has at least one amino acid whose replacement with respect to corresponding wild type polypeptide.A kind of preferred hK2 variant polypeptide comprises SEQ ID NO:8, promptly on 217 amino acids by the Xie Ansuan substituted lactamine.The antigenicity of hK2 polypeptide is identical with sophisticated hK2 molecule among Fig. 1, that is to say that described polypeptide also can be by can be with its specific combination but can not define with the antibody of hK3 (or hK1) cross reaction.Preferred described antibody capable reacts to each other with the antigen site or the epi-position that also are present in the ripe hK2 molecule of Fig. 1.

In series row number 08/096,946 (being U.S. Patent No. 5,516 at present, 639) in detail the antibody that can be used for defining the common antigen function has been described in detail, i.e. the polyclonal antiserum at hK2 subunit 41-56 of preparation in the body.

" isolating hK2 nucleotide sequence " is meant and contains more than 7, preferred more than 15, more preferably 20 or the RNA or the DNA of more a plurality of continuous nucleotide bases, described RNA or DNA are complementary to noncoding strand or the coding strand of natural hK2 polypeptide RNA or DNA, or can and can keep stable bond under stringent condition with their hybridization.Preferably, isolating nucleic acid encoding is a kind of bioactive hK2 polypeptide, its variant or its subunit.The method that the biological activated energy of hK2 polypeptide is understood thoroughly by this area detects, including, but not limited to the ability of hK2 polypeptid specificity antibody response, the ability (referring to embodiment 7) of cutting hK2 specific substrate, or with serum protein bonded ability (referring to embodiment 9).HK2 variant polypeptide or its subunit, or the subunit of hK2 polypeptide has the bioactive about at least 10% of the hK2 polypeptide that contains SEQ ID NO:1 aminoacid sequence, preferably about at least 50%, more preferably about at least 90%.

So, separated DNA or RNA be meant these nucleic acid at least not in its natural origin with the pollution of its nucleic acid that normally is connected, preferably basically without any other mammiferous DNA or RNA." do not have the pollution of the normal nucleic acid that connects at least " and comprise and the purpose nucleotide sequence imported in its source or the n cell again but be arranged in chromosomal different positions or its both sides are connected with this situation of the common non-existent nucleotide sequence of derived cell.The example of an isolating hK2 nucleic acid be coded have in bioactive hK2 polypeptide and the foregoing hK2 peptide have 90% identical RNA or the DNA of sequence at least with the homology zone (Fig. 1) that comes from hK3." separation, the purifying " that be used for the hK2 polypeptide obtains definition in following methodology discussion.

" recombinant nucleic acid " speech used herein, i.e. " recombinant DNA ", be meant from any and suitable tissue-derivedly derive or separate the nucleic acid that obtains, be DNA, it can change at external chemical method subsequently, import the purpose host cell then, as derive from the cell of animal, plant, insect, yeast, fungi or bacterium.The example of the recombinant DNA that obtains of " deriving " from a certain source is to be confirmed as encoding the useful segmental dna sequence dna of hK2 or its fragment or variant, and the mode that it can substantially pure is carried out chemosynthesis.From the example of this DNA of a certain source " separation " can be a kind of useful dna sequence dna, this dna sequence dna by chemical process (as utilizing restriction enzyme) cutting or from as described in take out the source so that can further process by genetic engineering method (as amplification) to be used for the present invention.

Like this, " recombinant DNA " comprises complete synthetic dna sequence dna, and semisynthetic dna sequence dna from biogenetic derivation separated DNA sequence, derives from the dna sequence dna of importing RNA and their mixture.In general, recombinant DNA sequence is not as the original composition in the host target cell of DNA acceptor, and perhaps it is arranged in its genome but does not express or be not high expression level.

" mosaic " used herein be meant that a carrier comprises and derive from least two DNA not of the same race, or comprise the DNA with a kind of source, but with in this kind under natural or wild-type status non-existent a kind of mode link together.

" regulating and controlling sequence " is meant and expresses the necessary dna sequence dna of the encoding sequence that can be operatively connected in a certain specific host organism.The regulating and controlling sequence that is applicable to prokaryotic cell prokaryocyte for example has promotor, selectively comprises operon sequence and ribosome bind site.The known genuine karyocyte can utilize promotor, polyadenylation signal and enhanser.

" can be operatively connected " and be meant nucleic acid placed with another nucleotide sequence that emic position is arranged.For example, if the dna sequence dna of coding presequence or secretion leading peptide is to participate in the excretory precursor protein matter formal representation of polypeptide, then it promptly is operationally to be connected with the DNA of these many skins; If promotor or enhanser influence transcribing of sequence, then be operationally to connect with this sequence; If translation is convenient in the ribosome bind site present position, then is to be operably connected with encoding sequence.In general, " can be operatively connected " is meant that the dna sequence dna of connection is a successive, and as in the leading peptide of secretion property, sequence continuously and be in and read in the frame.Yet enhanser does not need to be on the successive position.Connect at suitable restriction endonuclease sites and finish connection.If there is no such site utilizes synthetic oligonucleotide adapter or joint to carry out according to ordinary method.

" Southern analysis " or " Southern hybridization " is known with one and the oligonucleotide or the dna fragmentation that are labeled, determines at DNA or contains a kind of method that has some dna sequence dna in the restriction enzyme digestion product of composition of DNA by the hybridization means.Southern hybridization is usually included in electrophoretic separation DNA digestion product in the sepharose, denatured DNA after the electrophoretic separation, DNA is transferred on nitrocellulose filter, nylon membrane or other the suitable supporting film, save described method according to (source is the same) 9.37-9.52 such as Sambrook, analyze with the probe of radio-labeling, biotin labeling or enzyme labelling.

" Northern hybridization " or " Northern analysis " is to be used for determining energy and known probe, as oligonucleotide, and dna fragmentation, cDNA or its fragment, or a kind of method of the RNA sequence of RNA fragment hybridization.Probe by radio isotope (as ³²P), or vitamin H, or enzyme institute mark.RNA to be analyzed is electrophoretic separation in sepharose or polyacrylamide gel usually, transfer on nitrocellulose filter, nylon membrane or other suitable film, the method that adopts this area to understand thoroughly is hybridized method as described in saving as (source is the same) 7.39-7.52 such as Sambrook to it.

" stringent condition " is meant that (1) low ionic strength and height wash film temperature, as 0.015MNaCl/0.0015M Trisodium Citrate (SSC), 0.1% sodium lauryl sulphate (SDS), 50 ℃ of temperature, or (2) use denaturing agent in crossover process, as methane amide, as 50% methane amide/0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer (pH6.5), 750mM sodium-chlor, 75mM Trisodium Citrate, 42 ℃ of temperature.Another example is 50% methane amide, 5xSSC (0.75M sodium-chlor, 0.075M Trisodium Citrate), 50mM sodium phosphate (pH6.8), 0.1% trisodium phosphate, 5xDenhardt solution, through the salmon sperm DNA (50 μ g/ml) of ultrasonication, 0.1% sodium lauryl sulphate, 10% T 500,42 ℃ of temperature, washing is carried out in 0.2xSSC and 0.1%SDS at 42 ℃.Referring to other stringent condition in (source are the same) such as Sambrook for example.Expression cassette or expression vector

Comprising has been operably connected, and the expression cassette or the carrier of the recombinant DNA sequence of the coding of the promotor of function hK2 are arranged in host cell can be ring-type or linearity, strand or two strands.In general, expression cassette or expression vector are a kind of chimeric DNAs, and as plasmid DNA, it contains the control region of coding region and both sides, and this control region can promote recombinant DNA to express in the clone that produces.For example, expression cassette self may contain a promoters active in mammalian cell, maybe can utilize the promotor that has been present in the goal gene group that is transformed.These promotors comprise the CMV promotor, SV40 late promoter and retrovirus LTR (long terminal repetition element).Except the recombinant DNA sequence as the transcription unit of hK2 or its part, a part of recombinant DNA may not transcribed, and plays and regulates or structure function.

Expression cassette in the transfered cell or expression vector also comprise a selectable marker gene or reporter gene usually, and perhaps the both has, to make things convenient for from being determined the cell transformed group and the screening transformant.In addition, selective marker may be carried in another segment DNA, is used for the cotransformation process.Selective marker or reporter gene both sides all have suitable regulating and controlling sequence so that it is expressed in host cell.Know various effective selective markers in the art and comprise, for example microbiotic and herbicide resistance gene, as neo, hpt, dhfr, bar, aroA etc. are referring to the table 1 of people such as Lundquist (United States Patent (USP) NO.5,554,798).

The function that reporter gene can be used for identifying the potential transformant or estimates regulating and controlling sequence.The proteinic reporter gene that coding is analyzed is easily understood thoroughly in this area.In general, reporter gene is the gene that does not exist or do not express in receptor tissue or organism, can show the expression of its coded protein with some characteristics (as enzymic activity) that detect easily.Preferred gene comprises the chloramphenicol acetyl transferasegene (cat) that derives among the intestinal bacteria Tn9, the beta-Glucuronidase gene (gus) in intestinal bacteria uidA site, the luciferase gene in fluorescence worm Photinus pyralis source.The analysis report expression of gene can be carried out by the appropriate time after DNA imports recipient cell.

Other brings into play the element of function in host cell, as intron, and enhanser, polyadenylic acid sequences etc. also may become the part of recombinant DNA.These elements are not necessarily necessary element in DNA performance function, but may transcribe by influence, expression that the stability of mRNA etc. improves DNA.Can on demand these elements be introduced among the DNA and in cell, obtain optimal effectiveness in the hope of transfering DNA.

Structure commonly used is used to transform the method for the recombinant DNA of purpose cell and understands thoroughly in this area.Available same composition and construction process are set up the useful DNA of this paper.For example, J.Sambrook " molecular cloning: laboratory manual " (press of cold spring harbor laboratory, second edition, 1989) of writing provide suitable construction process.Transformed host cell also reclaims the hK2 recombinant polypeptide

Adopt transfection easily expression cassette or the expression vector that includes the recombinant DNA of coding hK2 polypeptide to be imported the purpose cell, as the calcium phosphate precipitation method of people such as C.Chen optimization in molecular cytobiology 7,2745 (1987).Transfection also useful commercial test kit (providing as BRL company) is undertaken by lipofection.

The host cell that is appropriate to express the hK2 polypeptide derives from multicellular organism.These cells have complicated processing and glycosylation activity.Say in principle, any higher eucaryotic cells culture, no matter it derives from vertebrates or invertebrates, all can be used among the present invention.The example of invertebral zooblast comprises plant and insect cell.Identified numerous baculovirus strains, mutant and the corresponding insect host cell that allows, as fall army worm (caterpillar), Egyptian her ant (mosquito), Aedes albopictus (mosquito), drosophila melanogaster (fruit bat) and silkworm.Referring to Luckow etc., biology/technology, 6,47 (1988); Miller etc., genetic engineering, volumes such as J.K.Settow, the 8th volume (PlerumPublishing, 1986), pp.277-279; Maeda etc., nature, 315,592 (1985).Many virus strain that are used for transfection can openly obtain, and as the Bm-5 strain of L-1 mutant strain and the silkworm NPV of autographa california NPV, these viruses preferably can be used for transfection fall army worm cell.

When the hK2 polypeptide was expressed in the reconstitution cell in non-human source, the hK2 polypeptide did not just contain protein or many skins of human origin fully.Yet, must be from recombinant cell protein matter or polypeptide the many skins of purifying hK2 to obtain the preparation of basic homogeneity in the hK2 polypeptide.For example, can remove cell debris, then separatory membrane and soluble protein part by centrifugal substratum or lysate.Can be from the soluble protein fraction purifying hK2 polypeptide, if necessary, can from the film fraction of cracking culture, carry out purifying.Subsequently can be from the soluble protein polluted or polypeptide purifying hK2 polypeptide, adoptable method has the affine or ion exchange column separation of immunity, ethanol sedimentation, reversed-phase HPLC, silica gel column chromatography or anion exchange resin layer are analysed (as DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel-filtration (as utilizing Sephadex G-75), or part affinity chromatography.

In case from the gained genetically modified host cell, separate, just be easy to prepare the derivative and the variant of hK2 polypeptide.As the method that carboxyl or precursor conversion is become acid amides that adopts that this area understands thoroughly, also can prepare the acid amides of hK2 polypeptide of the present invention.A kind of preferred method of C-terminal Carboxylamideization be from solid support with suitable amine cracking polypeptide, perhaps under the condition that alcohol exists, carry out cracking, generate ester, carry out amino Decomposition with suitable amine subsequently.

The carboxyl salt of the many skins of preparation hK2 can be mixed peptide and carry out according to a conventional method with one or how normal purpose alkali, described alkali for example is metal hydroxides alkali (as sodium hydroxide), metal carbonate or supercarbonate (as yellow soda ash or sodium bicarbonate); Or amido alkali, as triethylamine, trolamine etc.

The preparation of the N-acyl derivative of this polypeptide amino can be carried out last condensation reaction by the amino acid with the protection of N-acyl group, or protection or unprotected peptide acidylate are finished.Preparation O-acyl derivative can be finished by the acidylate that for example makes free hydroxyl peptide or peptide resin.Two kinds of acidylates all can adopt conventional acylating reagent to finish, as acyl halide, and acid anhydride, acylimidazole etc.As needs, O-acidylate and N-acidylate can be carried out simultaneously.In addition, the internal amino acid sequence of hK2 can be carried out the substituting of 1 or 2 conserved amino acid (comprise with the D type but not L type amino acid substitute) and modified at specified location among Fig. 1.

Be the acidify salt of preparation polypeptide, can in polypeptide, add one or how normal mineral acid or organic acid (example hydrochloric acid) and carry out.The carboxyl ester of polypeptide also can be undertaken by the common method that this area is understood thoroughly.The hK2 polypeptide variants

Have realized that the hK2 polypeptide variants with respect to SEQ ID NO:1, SEQ ID NO:3 or SEQID NO:5 have 1 amino acid whose replacement at least, and as relative SEQ ID NO:1, SEQ ID NO:8 has by the replacement of L-Ala to Xie Ansuan at 217.Especially, amino acid whose replacement is carried out in a kind of conservative relatively mode.In table 1, this conservative replacement is presented at the typical case and replaces under the title, and preferred replacement is shown under the title of preferred replacement.After introduce replacing, the biological activity of examination product, as producing the ability of hK2 specific antibody, or with the ability of hK2 specific antibody (promptly combine and not with hK3 (PSA) bonded antibody) specific reaction with hK2.

The original residue typical case of table 1 replaces preferred L-Ala (A) Xie Ansuan of replacing, leucine, Isoleucine Xie Ansuan arginine (R) Methionin, glutamine, l-asparagine Methionin l-asparagine (N) glutamine, Histidine, Methionin, arginine glutamine aspartic acid (D) L-glutamic acid L-glutamic acid halfcystine (C) Serine Serine glutamine (Q) l-asparagine l-asparagine L-glutamic acid (E) aspartic acid aspartic acid glycine (G) proline(Pro) proline(Pro) Histidine (H) l-asparagine, glutamine, Methionin, arginine arginine Isoleucine (I) leucine, Xie Ansuan, methionine(Met), L-Ala

Phenylalanine, nor-leucine leucine leucine (L) nor-leucine, Isoleucine, Xie Ansuan is used methyllanthionine,

L-Ala, phenylalanine Isoleucine Methionin (K) arginine, glutamine, l-asparagine arginine methionine(Met) (M) leucine, phenylalanine, Isoleucine leucine phenylalanine (F) leucine, Xie Ansuan, Isoleucine, L-Ala leucine proline(Pro) (P) glycine glycine Serine (S) Threonine Threonine Threonine (T) Serine Serine tryptophane (W) tyrosine tyrosine tyrosine (Y) tryptophane, phenylalanine, Threonine, Serine phenylalanine Xie Ansuan (V) Isoleucine, leucine, methionine(Met), phenylalanine, leucine

The L-Ala nor-leucine

Amino acid in the scope of the invention is replaced, and generally select for use those can significantly not change them in the replacement of keeping aspect following: (a) polypeptide backbone is in the structure of replacing the zone, as lamella or helical conformation; (b) molecule is in the electric charge and the hydrophobicity of target site; (c) size of side chain.Character according to side chain is divided into following several groups with naturally occurring amino acid: (1) hydrophobicity: nor-leucine, methionine(Met), L-Ala, Xie Ansuan, leucine, Isoleucine; (2) neutral hydrophilic: halfcystine, Serine, Threonine; (3) acidity: aspartic acid, L-glutamic acid; (4) alkalescence: l-asparagine, glutamine, Histidine, Methionin, arginine; (5) influence the residue that chain moves towards: glycine, proline(Pro); (6) aromatic series: tryptophane, tyrosine, phenylalanine.

The present invention also comprises the hK2 variant of non-conservative replacement.A member during non-conservative replacement comprises one of above-mentioned group changes into the member in another group.With dna molecular among amino acid replacement introducing the present invention is to be undertaken by the method that this area is understood thoroughly.The purposes of reorganization hK2 polypeptide

In case separate, can and the variant of antigenic activity be arranged with the hK2 polypeptide, derivative and fragment are used for carrying out hK2 and analyzing deriving from the sample of suspecting the biomaterial that contains hK2 or anti-hK2 antibody, as US Patent No 5,516, described in 639.For example, by as one or more radiolabeled peptide residue, can be with detectable sign on the hK2 polypeptide marker, be used for competing in conjunction with anti-hK2 antibody with endogenous hK2, promptly as the anti-hK2 antibodies in " capturing antigen " and the physiological liquid sample, with the combining of hK2 competition and fixable anti-hK2 antibody, step is as follows by multiple competition immunoassay mode:

(a) provide a certain amount of anti-hK2 antibody that is attached to solid surface;

(b) mixing contains the physiologically sample of hK2 and the hK2 polypeptide that has detectable label of known quantity, to form mixing sample;

(c) described antibody and described mixing sample are fully acted on, make between antibody and hK2 immune response takes place, generate a kind of antibody-hK2 mixture, and immune response takes place between antibody and the many skins of described mark to form antibody-labeling polypeptide mixture.

(d) separation reaches and labeling polypeptide bonded antibody with hK2 bonded antibody from mixing sample;

(e) detect or measure and be attached to the labeling polypeptide of antibodies of solid surface and existence or the amount that still is present in the labeling polypeptide in the mixing sample;

(f) determine existence or the quantity of hK2 the sample from the result of step (e).

Suppressing in the another kind of method of endogenous hK2 in the immunoassay test sample, in the sample that contains the endogenous hK2 of unknown quantity, add the anti-hK2 antibody of known quantity by competition.Select to add the amount of antibody, make it than all lacking in conjunction with the required amount of hK2 (as the amount that in the specimen material of the same quantity of patients with prostate cancer, may exist) of estimating existence in the sample.Then, add wherein being marked with among the present invention of known quantity of detectable label hK2 polypeptide or its subunit.If endogenous hK2 is present in the sample, so seldom some antibody combine with the hK2 polypeptide of mark, and it will exist in solution with free state like this; If no endogenous hK2 in the sample, the labeling polypeptide of Jia Ruing will form the divalence mixture with anti-hK2 antibody response so.IgG antibody with resisting mammal (sheep, goat, mouse etc.) precipitates the antigen-antibody complex of divalence subsequently.The amount of endogenous hK2 is inversely proportional in the middle radioactivity of throw out (trivalent mixture) or other labelled amount and the sample.For example, the reduction of radioactivity means the existence of endogenous hK2 in the precipitation.

Except preparation polyclonal antibody or sero-fast ordinary method in laboratory animal and domestic animal, can utilize known Hybridoma Cell Culture technology to prepare the monoclonal antibody of anti-hK2 polypeptide.In general, this method comprises that preparation can produce the fused cell system of antibody, as the fusion of elementary splenocyte and compatible myelomatosis continuous cell line, by a large amount of culture techniques of cell or change the myeloma cell over to and originate and cultivate in animal or the compatible animal strain, make the fused cell growth.Compare with the antibody that the inoculation animal produces, this kind antibody has more advantage, and for example they have excellent specificity, susceptibility, and on immunochemistry relative " pure ".The immunocompetence fragment of these antibody such as F (ab) fragment and the humanized monoclonal antibody of part also belong within the scope of the present invention.Chimeric and modified antibodies

Chimeric antibody comprises from the fusion between the constant region in the variable region in a kind of immunoglobulin (Ig) source and another kind of immunoglobulin (Ig) source.The variable region derives from the immunoglobulin (Ig) of another kind usually, perhaps is the people.This technology is well-known in the art.Referring to for example European patent application EP-A-0125 023 (Cabilly/Genetech) and EP-A-0120694 and US Patent No 4,816,567.The variant for preparing the immunoglobulin (Ig) quasi-molecule with recombinant DNA method is disclosed in these documents.

Another kind of preparation approach chimeric or modified antibodies is that the variable region with monoclonal antibody is connected generation chimeric molecule derivative (referring to WO86/01533, Neuberger and Rabbits/Celltech) with another NIg molecule.Another kind method is a kind of gomphosis immunoglobulin of preparation, makes it have different specificity (referring to EP68763A) in different variable regions.Also having a kind of method is to introduce a sudden change in the DNA of coding monoclonal antibody, is not changing some character that changes it under its specific substantially situation.This can finish by other technology of being understood thoroughly in site-directed mutagenesis or this area.

Winter patent application EP-A-0239400 has disclosed and has utilized recombinant DNA technology (" CDR transplanting ") the preparation method of the derived antibody of change to some extent, promptly uses the not complement determining area (CDR) of the alternative immune globulin variable region of complement determining area of homospecific immunoglobulin (Ig).Like this, combine with the change of ramework region, the CDR implantation technique can make the antibody humanization.

Also can rebuild human immunity system in the mouse body that lacks natural immune system, immune then this mouse is with the generation human antibodies special to immunogen, thus preparation people antibody.

Comprising the humanized antibody to the rodents antibody CDR zone of antigen-specific interested, is not the dissident by the human immunity system identification more likely.Therefore, have the specific humanized antibody of identical combination,, may in human gene therapy and/or diagnosis, special purposes be arranged as HK1G464.

Operation and/or change the gene of any known antibodies or this antibody of encoding is with the derive technology of antibody of generation, by being understood thoroughly this area.Utilize reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the special transcript of hK2

In order to detect the rna transcription thing of coding hK2, from suspection isolation of RNA the cell sample of hK2 RNA is arranged, as from human prostate tissue, separating total RNA.The method isolation of RNA that can adopt this area to understand thoroughly, as utilize TRIZOLTM reagent (GIBCO-BRL/Life Techno1ies).Can utilize Oligo-dT to prepare the first chain cDNA as the primer in the ThermoScript II reaction from isolating RNA, cDNA first chain of generation increases in the PCR reaction subsequently.

" polymerase chain reaction " or PCR are meant process or the technology of nucleic acid fragment, DNA and/or RNA that amplification is a large amount of selected, and this technology is in US Patent No 4,683, are described in detail in 195.Usually, according to the sequence information design oligonucleotides primer of area-of-interest two ends or both sides.These primers are same or similar on sequence with the relative chain of template to be amplified.The RNA sequence that available pcr amplification is special, the specific DNA sequence in total genomic dna source, from the cDNA that total cell RNA is transcribed, phage or plasmid sequence etc.Generally referring to Mullis etc., the port symposial of hydrobiology cold spring, 51,263 (1987); Erlich etc., round pcr (StocktonPress, NY, 1989).Like this, the amplification of specific nucleic acid sequence being carried out with PCR depends on oligonucleotide or the primer that contains conservative nucleotide sequence, can be by genes involved or protein sequence as relatively inferring this conserved sequence with the sequence of Mammals hK2 gene.For example, expectation of preparation can with the antisense strand annealed primer of the DNA of coding hK2 polypeptide and expectation can with positive-sense strand annealed primer.

Usually, isolating RNA cooperates with a primer in ThermoScript II (RT) reaction, produces strand cDNA.O1igo-dT, random sequence oligonucleotides and sequence-specific oligonucleotide all can be used as primer in reverse transcription reaction.Referring to (source are the same) such as Sambrook.Produce amplified production with sequence specific primer amplification strand cDNA subsequently.

For detecting amplified production, usually reaction mixture is carried out agarose gel electrophoresis or other simple and easy isolation technique, and whether the special amplified production of detection hK2 exists.The hK2DNA that detects amplification also can be undertaken by following method: fragment is cut from glue or eluted (for example, referring to Lawn etc., nucleic acids research, 9,6103 (1981), Goeddel, nucleic acids research, 8,4057 (1980)), extension amplification outcome is arrived suitable carrier (as pCR II carrier, cloning site Invitrogen), measure the clone and insert fragments sequence, and the known array of this dna sequence dna and hK2 is compared.In addition, the special oligonucleotide probe of also available hK2 detects the DNA amplification of hK2 by for example Southern hybridization, or with the DNA standard of the known molecular amount electrophoretic mobility of DNA amplification relatively.

Embodiment with reference to following detailed description further describes the present invention.The structure of embodiment 1 materials and methods Mammals hK2 expression vector

Adopt the special Oligonucleolide primers of a pair of hK2 (5 ' ACGCGGATCCAGCATGTGGGACCTGGTTCTCT 3 '; SEQ ID NO:9 and 5 ' ACAGCTGCAGTTTACTAGAGGTAGGGGTGGGAC 3 '; SEQ ID NO:10). (RT-PCR) synthesizes a cDNA who is about to 820bp to the RNA that organizes from people BPH through reverse transcription-polymerase chain reaction, as shown in Figure 2, former hK2 (pphK2) before this cDNA coding is whole is with respect to the 40-858 position Nucleotide of pphK2 transcript initiation site.The both sides of 5 ' and the 3 ' end (with respect to the positive-sense strand of pphK2) of this cDNA have BamH I and Pst I restriction enzyme site sequence respectively.Subsequently, cDNA is through the agarose gel electrophoresis purifying, and BamH I and Pst I enzyme are cut digestion.CDNA through digesting is connected with the pVL1393 plasmid vector of cutting through BamH I-Pstl enzyme and is transformed in the intestinal bacteria HB101 bacterial strain.Screening contains the intestinal bacteria of pphK2 cDNA/pVL1393 plasmid vector, measures the insertion fragments sequence that it contains pphK2.A large amount of plasmid pphK2 cDNA/pVL1393 that cultivate are also with CsC1 gradient ultracentrifugation purifying in intestinal bacteria.

According to Budapest clause, the intestinal bacteria HB101 that will contain plasmid pphK2/pVL1393 on May 2nd, 1994 is preserved in American type culture collection, and the registration number of distribution is ATCC69614.

(give with the plasmid pVL1393 that contains pphK2 by doctor Young, Mayo Clinic) is template, utilize primer: A (5 ' ATATGGATCCATATGTCAGCATGTGGGACCTGGTTCTCTCCA3 ') (SEQ ID NO:11) and B (5 ' ATATGGATCCTCAGGGGTTGGCTGCGATGGT3 ') (SEQ ID NO:12) obtain representing the 0.8Kb fragment (Fig. 2) of whole pphK2 coding region by pcr amplification.With the PCR product insert the TA-clonal antibody (Invitrogen Corp., San Diego, CA) in and measure the segmental dna sequence dna of whole insertion.

In order to obtain Mammals hK2 expression vector, pGT-d plasmid (Berg etc. are separated and be inserted into to the insertion fragment that will contain hK2 from corresponding TA clone, nucleic acids research, 20,54-85,1992) (given by Brian Grinnell, Lilly) the Bcl I site in is positioned under the GBMT promoter regulation.The 0.8Kb wild-type hK2 that derives from corresponding TA carrier is inserted fragment be cloned into (Miller etc., biotechnology, 9,980 (1989)) among plasmid pLNSX and the pLNCX respectively, express carrying agent PLNS-hK2 and PLNC-hK2 thereby obtain Mammals.Inserting the direction of fragment in all mammalian expression vectors is identified by dna sequencing.The generation of recombinant clone

AV12-664 (ATCC CRL-9595) is a clone that derives from through adenovirus inductive Syria hamster, the Du145 cell cultures is in DMEM (high glucose) substratum that has replenished 10% foetal calf serum (D10F), and the PC3 cell cultures is in containing 10% foetal calf serum MEM substratum.With calcium phosphate method (Maniatis etc., the source is the same, 1989) with the transfection of hK2 expression vector in the AV12 cell, transfection was resuspended in cell in the D10F+200nM methotrexate (MTX) after 3 days.2-3 separates drug-fast cell clone after week, collect the consumption substratum and carry out the Western analysis.With lipofection amine reagent (Gibco-BRL, Gaithersburg, MD) with the transfection of hK2 mammalian expression vector in PC3 and Du145, screening obtains PC3-hK2 and DU145-hK2 clone in the substratum that contains 400 μ g/ml G418.Protein purification and order-checking

AV12-hK2 clone is cultivated in D10F+200nM MTX substratum, and cell grows to about 60% when merging, and with Hanks balanced salt solution washed cell, and it is resuspended in the HH4 substratum of serum-free.The consumption substratum that adds serum-free is collected the consumption substratum and is stored in-20 ℃ after 7 days.For protein purification, the consumption substratum of serum-free is concentrated, change the 50mM sodium hydrogen carbonate solution of pH8 into.Sample directly pumps into TSK DEAE-5PW HPLC post after 0.2 μ filter filters, 21mm * 150mm, flow velocity are 5ml/ minute.The 50mM sodium bicarbonate that contains pH7.9 in the buffer A, buffer B contain 50mM sodium bicarbonate and 0.5M sodium-chlor, pH7.6.Obtain elution curve with following gradient elution: the buffer B of 0-50% 35 minutes, 35-40 minute is the buffer B of 50-100%, 100% buffer B isocratic elution 5 minutes carries out balance again with buffer A then.Flow velocity kept 5ml/ minute always.In above step, available borate buffer solution replaces bicarbonate buffer, can't produce remarkably influenced.

Adopt dry ELISA method (as follows), with exist (Saedi etc., molecular cell incretology, 109,237 (1995)) of hK2 in the anti-pphK2 antibody test of the rabbit DEAE fraction.Compiling has active each fraction of hK2, through ultrafiltration it is concentrated into about 5-8ml with film (10KD molecular weight cut-off), adds solid ammonium sulfate to final concentration 1.2M.Sample is to PolyLC polypropylene asparagine post on the sample, aperture 1000A, and 4.6mmX200mm is with by hydrophobic interaction chromatography (HIC) isolated protein.Buffer A contains the 20mM sodium phosphate, 1.2M sodium sulfate, pH6.3; Buffer B contains the 50mM sodium phosphate, 5% Virahol, pH7.4.Gradient is 0-20% buffer B 5 minutes, 5-20 minute is the 20-55% buffer B, 20-23 minute is 55% buffer B isocratic elution, 23-25 minute is the buffer B wash-out of 55-100%, 100% buffer B isocratic elution is 2 minutes again, buffer A weighs balance, flow velocity 1ml/ minute then.The HIC elution peak that contains hK2 elutes in about 50% buffer B, changes in the borate buffer solution of 50mM (pH8) after Centricon-10 (Amicon) (molecular weight cut-off 10KD) ultrafiltration repeats to concentrate.Measure its purity with SDS-PAGE and Western hybridization analysis.Be used to estimate hK2 ^V217The optical extinction coefficient of concentration is 1.84A ₂₈₀=1mg/ml.

Under some situation, the HIC peak that contains hK2 further uses 10/30 Pharmacia S12 post to carry out purifying through size exclusion chromatography (SEC).In this purifying, contain the HIC peak of hK2 such as above-mentioned ultrafiltration and be concentrated into less than 1ml, go up sample then on the sodium borate buffer liquid equilibrated size exclusion chromatography post of the ammonium acetate of 100mM pH7 or pH8.Flow velocity is 0.7ml/ minute, subsequently with hK2 peak ultrafiltration and concentration.Damping fluid is removed through lyophilize in the peak in ammonium acetate that will collect from the size exclusion chromatography post, and is heavy more soluble in water.Get a part of sample in hydrolysis 20 hours in gasiform 6N hydrochloric acid under 112 ℃ of vacuum states, heavily be dissolved in the 0.1N hydrochloric acid again, analyze with Hewlett Packard Amino quant amino acidanalyser, with OPA primary amine is carried out the effect of amino acid pre-column derivatization, secondary amine is carried out the effect of amino acid pre-column derivatization with MOC.

With the affine resin of HK1G586.1 (as follows) hK2 is carried out affinitive layer purification.According to Schneider (journal of biological chemistry, 257,10766 (1982)) method combines HK1G586.1 monoclonal antibody (mAb) with Pharmacia GammaBind Plus Sepharose (cat.No.17-0886), be summarized as follows: HK1G586.1 monoclonal antibody and resin are rotated to stir under 4 ℃ of conditions spend the night.To resin centrifugal (500xg 5 minutes, 4 ℃), use 0.2M trolamine (pH8.2) washing resin 2 times.Under room temperature (22 ℃) condition, in the linking agent (0.2M trolamine, the 25mM dimethyl pimelimidatedihydrochloride among the pH8.2) of new preparation with crosslinked 45 minutes of amido.Cancellation resin 5 minutes under the room temperature in the thanomin of 20mM pH8.2, back are with the phosphate radical of 1M sodium-chlor, 0.1M, and pH7.0 washing 2 times is used the phosphate buffered saline(PBS) washing resin 2 times again, is stored among the 0.05%NaN3 standby in 4 ℃.

Adopt Applied Biosystems Model 477a pulse liquid-phase sequencing instrument that protein and peptide are checked order.477a type sequenator is used the automatic edman degradation chemistry, and amino acid is discharged from the N-terminal order, forms the PTH derivative subsequently and carries out stratographic analysis by reversed-phase HPLC.The peptide sample is splined on sequenator on the glass fiber filter upholder that biobrene handles, the holoprotein sample is splined on the filter membrane that biobrene handles, or be splined in advance (Beckman on the activated Porton filter membrane, Fullerton, CA), the sample trace that sequenator gets off is at first at the (Novex of Novex system, San Diego, CA) carry out electrophoresis with microgel in, go to then on the Problot pvdf membrane, with Xylene Brilliant Cyanine G colour developing, directly check order after downcutting suitable band from pvdf membrane.MONOCLONAL ANTIBODIES SPECIFIC FOR

First day will be in complete freund adjuvant (CFA) 50 μ l phK2 peritoneal injection A/J mouse, the 14th day phK2 25 μ gs of peritoneal injection in incomplete freund adjuvant, and be dissolved in the phK2 25 μ g of PBS at the 28th day peritoneal injection.Merged before 3 days, the phK2 intravenously that is dissolved in PBS with 10 μ g is strengthened mouse immune.After killing mouse, make single cell suspension by spleen.Bone-marrow-derived lymphocyte and myeloma cell P3.653 are merged, with ELISA screening and cloning hybridoma, and according to it to hK2 ^V217Go up cleer and peaceful phK2 ^V217Supernatant reactive and select with the minimal reaction of PSA.Obtain 2 clone HK1G464 and HK1G586 according to these standards, with FACstar pulse cell sorter single hybridoma is assigned to and carries out subclone on the mice spleen feeder layer.Subclone HK1G464.3 and HK1G586.1 are used to further analysis.

Except immunogen is hK2 ^V217, with alum replaced C FA and IFA, replace adopting step same as described above to carry out another fusion outside the A/J mouse with BALB/C mice, obtain cloning HK1H247.

According to budapest treaty, hybridoma is cloned HK1G464.3, HK1G586.1, HK1H247, HK1A523 and HKD106.4 are preserved in American type culture collection, and registration number is respectively HB11983, HB12026, HB12162, HB11876 and HB11937.The mono-clonal of hK2 peptide based immunogens and Polyclonal Antibody Preparation

100 μ g KLH coupling peptide subcutaneous vaccination sheep and goats that will be in complete freund adjuvant (CFA), are used for 100 μ g peptide booster immunizations of incomplete freund adjuvant (IFA) at 3 weeks at interval.

Be the monoclonal antibody of preparation peptide based immunogens,, three weeks at interval, be used for 100 μ g peptide booster immunizations of incomplete freund adjuvant the KLH link coupled peptide subcutaneous vaccination immunity Balb/c mouse of 100 μ g in complete freund adjuvant (CFA).Perhaps, with 50 μ g couplings the peptide peritoneal injection immunity A/J mouse secondary of KLH, to the mouse of positive titre once with 25 μ g coupling agent intravenously booster immunizations.

After first three time immunity, got animal blood antagonist in 6-10 days after the immunization and test.Peptide is fixed on 0.25 inch polystyrene beads (Clifton, Clifton Heights, PA), on average each globule and 1 μ g bovine serum albumin link coupled peptide in the carbonate buffer solution of pH9.6 4 ℃ be incubated overnight.With the 0.01M phosphate buffered saline(PBS) that contains 0.1% polysorbas20, pH7.4 (PBS) washing globule 3 times seals with 1% skim-milk and 1%BSA.With globule and 250 μ l 1: 100,1: 1000 or 1: 10000 common incubation of dilution animal serum 18 hours.After washing 3 times, every globule with horseradish peroxidase (Cappel-OrganonTeknica Corp., Durham, Nc) the anti-sheep of link coupled 250 μ l rabbits, anti-mouse of rabbit or the anti-goat antibody of rabbit are with 150rpm incubation 3 hours on horizontal incubator.With the O-Phenylene Diamine is substrate, with spectrophotometer detection by quantitative enzyme signal.Make negative control with non-immune serum, and represent the observed value of immune serum with the multiple of contrast reading.

Will be from the mice spleen of the serum titer that is positive isolating lymphocyte and myeloma cell merge to produce hybridoma.With aforesaid method the antibody that these cell clones produce is screened.With limiting dilution assay positive colony is carried out subclone and screening again.The intraperitoneal that the mono-clonal hybridoma is expelled to mouse originally is to produce ascites.

For purifying mono-clonal and polyclonal antiserum, at first carry out the molecular size chromatography and antagonistic Serum carries out the IgG separation with saturated ammonium sulphate and super gel ACA-34 post.Utilization is by being coupled to the further affinitive layer purification polyclonal antibody of post that Sepharose 4B makes with peptide through cyanogen bromide.With acid PBS (pH2.45) antibody purification is eluted from post.Elisa assay

Using dry elisa assay measures these clones' serum-free and consumes the hK2 that collects in hK2 in the substratum and the hK2 purge process in the fraction.Under 37 ℃, (Becton Dickinson Labware NJ) spends the night to cover titer plate with 50 μ l consumption substratum or post fraction.Wash each hole with the PBS that contains 0.1% polysorbas20 (PBST), and educated 1 hour with 50 μ l, one temperature resistance.Wash culture hole once more with PBST, add coupling have horseradish peroxidase (1: 500, JacksonImmunosearch Laboratories, Inc., West Grove, 50 μ l sheep anti-mouse iggs PA) or goat anti-rabbit igg-Fc antibody were 37 ℃ of incubations 1 hour.Wash culture hole with PBST, (MO) incubation is 5 minutes for OPD, Sigma, and (the EL310 type VT) is measured the reading of color reaction at A490 for Biotek Instruments, Inc. with the ELISA readout instrument with the O-Phenylene Diamine dihydrochloride.All samples is all analyzed secondary, with transfection the consumption substratum of AV12 cell of empty plasmid make negative control.

In liquid phase ELISA, available biotinylated phK2 ^V217, hK2 ^V217Test with the PSA antagonist.Method purifying PSA according to Sensabaugh and Blake (urology magazine, 144,1523 (1994)).To be diluted in 50 μ l buffer A (8.82mM citric acid, 82.1mM sodium phosphate (diatomic base), 10% bovine serum albumin, 0.1% mannitol, 0.1%Nonidet P-40, pH7.0) the biotinylated hK2 of the 20ng in ^V217Or phK2 ^V217, perhaps being diluted in 0.25ng biotinylation PSA and 50 μ l hybridoma supernatants among the PBS that contains 10% horse serum (HS), the negative control supernatant is (that is, for phK2 ^V217And hK2 ^V217, for irrelevant hybridoma supernatant, for PSA, be the irrelevant 20 μ g/ml monoclonal antibody purifications that are dissolved in the horse serum), or the positive control supernatant (that is, be that 20 μ g/ml are dissolved in the monoclonal antibody purification PSM773 (anti-PSA) in the horse serum for PSA, or for phK2 ^V217And hK2 ^V217Supernatant for hybridoma HK1D104 (anti-hK2)) common incubation.The monoclonal antibody HC0514 of a kind of anti-hCG is used as negative control in PSA analyzes, in hK2 analyzed, the monoclonal antibody ZTG085 of a kind of anti-tau was as negative control.

With these antibody and antigenic mixture the titer plate of streptavidin bag quilt (Labsystems, Helsinki, Finland) in incubation 1 hour, constantly vibration.The PBS (PBST) that contains 0.1% polysorbas20 with 300 μ l washes titer plate three times, γ specificity mountain sheep anti-mouse igg antibody (Jackson Immuno Research Laboratories with the 100 μ l horseradish peroxidase that are diluted in HS at 1: 10000, Inc., Westgrove, PA) the vibration incubation is 1 hour.With after the PBST flushing, add 100 μ l for the second time, 0.03% sodium perborate, pH5.0 (SigmaChemical, St.Louis, MO) the 1mg/ml O-Phenylene Diamine in, vibration colour developing 30 minutes in 50mM phosphate-citrate salts damping fluid.Add 50 μ l 4N sulfuric acid termination reactions.The photoabsorption of measuring 490nm and 540nm place with the titer plate reader is to determine colour intensity.The 490nm absorption value surpasses at 2.6 o'clock and revises with the reading at 540nm place.The sample measurement value is the standard deviation of mean number ± three time measurement, and control value is the mean number that secondary is measured.The Western hybridization analysis

Adopt standard step to carry out the Western hybridization analysis.With Centricon10 (AmiconInc., Beverly MA) concentrate 10 times with the consumption substratum of serum-free, in 12% gel, carry out the SDS/PAGE electrophoresis (Bio-Rad Inc, Melville, NY).For analysis purposes, on the 8-25% gradient glue, carry out the SDS/PAGE electrophoresis with Pharmacia PhastSystem.Electrophoresis moves on to protein transduction on the nitrocellulose filter after finishing, and spends the night with the sealing of 2% skim-milk among the PBS under 4 ℃ of conditions.Hybond membrane resists the (ascites of dilution in 1: 1000 with one after washing, the perhaps monoclonal antibody or the polyclonal antibody of 1 μ g/ml purifying) 22 ℃ of following incubations 1 hour, wash then Hybond membrane and with two anti-(coupling of dilution in 1: 500 the goat anti-mouse antibody of horseradish peroxidase or coupling the goat anti-rabbit antibodies of horseradish peroxidase, JacksonImmunosearch Laboratories, Inc., West Grove, PA) incubation.(Sigma, St.Louis MO) and hydrogen peroxide, or press operational manual and use the ECL system (Amersham, Buckinghamshire England) make the colour developing of hybridization band, thereby detect the immunoreactivity band with DAB.The formation of covalent complex

Be the formation of test covalent compound, with the inhibitor of the hK2 of 0.175 μ M and 20 μ M at incubation under the pH8 condition in the 100mM borate buffer solution.The inhibitor of test comprises the 1-chymotrypsin inhibitor, 1-antitrypsin, 1-antiplasmin, antithrombin and 2-macroglobulin.In 5 μ l hK2 (10 μ g/ml), add the calculated amount inhibitor that is prepared in the 100mM borate buffer solution, if necessary, the cumulative volume of every duplicate samples can be adjusted to 10 μ l.Sample adds the 7xPhastSystem SDS sample buffer that 1.5 μ l contain 35% beta-mercaptoethanol 37 ℃ of incubations 3 hours, and water-bath was boiled sample 3 minutes.Before sample carries out SDS/PAGE and Western hybridization, sample is diluted in the SDS sample buffer at 1: 4.The proteolysis of peptide substrates

In order to detect the ability of hK2 cutting peptide substrates, the concentration of peptide with 10mg/ml is dissolved in the dimethyl sulfoxide (DMSO) (DMSO), the borate buffer solution with 100mM pH8 carries out dilution in 1: 10 then, contains PSA in this damping fluid, hK2 or trypsinase.Typical experimental procedure is as follows: 1 μ l peptide is added in the 7 μ l 100mM borate buffer solutions, add 2 μ l hK2 (10 μ g/ml) subsequently, PSA (500 μ g/ml), or trypsin 0.5 μ g/ml).Usually sample was 37 ℃ of incubations 16 hours.

With 100 μ l 0.2%TFA/ water inactivation samples, directly be splined in the Vydac C-18 reversed-phase column, this post is connected with the Biorad Model 800HPLC that disposes the automatic injector of AS100, two 1350 pumps and Biodimension scan-type UV-VIS probe.Solution A is a 0.1%TFA/ water, and solution B is the acetonitrile that contains 0.1%TFA.Sample in 90% solvent orange 2 A on sample, in 10 minutes, gradient becomes 60% solvent B.Simultaneously in 220nm and 280nm place monitoring absorption value.

Concentrate the collected peak of HPLC with traditional vacuum or lyophilize, go up subsequently sample in the Protein Sequencer with definite each fragment.Sometimes the deactivation sample of 10 μ l is directly gone up sample to the order-checking film, and, because sequence is known, so distribute to determine cleavage site by each amino acids in circulation.Carry out the proteolytic enzyme analysis with chromogenic substrate

The detection of p-nitrophenyl amine derivative substrate hydrolysis is to carry out with the HP8452A UV-VIS spectrophotometer of being furnished with able to programme and heat-staple 7 Room carriages.Test in 37 ℃ 100mM dobell's solution pH8 at incubation, measure its absorption intensity increased value at 405nm.Methoxyl group succinyl--Arg-Pro-tyrosine-p-Nitroaniline (MeO-Suc-R-P-Y-pNA) and the concentration of H-D-proline(Pro)-phenylalanine-arginine-p-Nitroaniline (P-F-R-pNA) in test are 1mM.

Listed peptide all except that No. 2 and No. 5 among the synthetic Figure 16 of the ABI 431A type peptide synthesizer of employing standard FastMoc chemistry, No. 2 is angiotensinogen and No. 5 oxidized form Regular Insulin β chains, all available from Sigma.Determine the molecular weight of each synthetic peptide chain with mass spectrograph by ES/MS, and confirm peptide sequence with above-mentioned ABI 477a type sequenator.PhK2 ^V217To hK2 ^V217Conversion

Will be in the 100-400 μ g/ml phK2 in the 50mM Sodium Tetraborate ^V217Sample is at 37 ℃ of following and 1% (weight ratio) trypsinase or the common incubations of hK2.Body protein is to the conversion process of mature protein in the past in monitoring, and this is by getting the hK2 of 1-2 μ g ^V217Parent material is diluted in the 100 μ l HIC damping fluids, with HIC-HPLC 2 kinds of forms is separately carried out as mentioned above.Except that shown in Figure 17 B, quantity being had these two kinds of forms while incubations of comparability, carry out hK2 with the same manner ^V217And phK2 ^V217Incubation.HK2 in example 2 mammalian cells ^V217Expression and purification

In order to express hK2 in mammal cell line, with PCR method hK2 (pphK2) the whole encoding sequence 0.8Kb fragments (Fig. 2) that increase, subclone is in PCRII (TA) carrier and be separated to several clones.Mensuration wherein several clones' whole pphK2 is inserted segmental nucleotide sequence, may be by the sudden change of pcr amplification generation to detect.

Select two clones and do further to analyze, wherein clone TA-hK2 and contain wild-type hK2 insertion fragment, another clones TA-hK2 ^V217The hK2 that contains sudden change inserts fragment.TA-hK2 ^V217650 bit codons at hK2 contain the replacement of a C to T, cause the amino-acid residue generation L-Ala (GCT) of 217 of hK2 to replace (Fig. 2) to the conservative type of Xie Ansuan (GTT).In order to obtain mammalian expression vector, with TA-hK2 and TA-hK2 ^V217PphK2 insert the fragment subclone in the PGT-d plasmid, they are under the control of GBMT promotor, thereby obtain pGThK2 and pGThK2 ^V217Plasmid (Fig. 3).The GBMT promotor comprises several regulating and controlling sequences, and can be activated (Berg etc., the source is the same, (1992)) by adenovirus Ela protein.

In order to detect pphK2 ^V217Can gene product be expressed in mammalian cell, with plasmid pGThK2 ^V217Transfection is in the AV12-664 cell.This clone derives from the tumour of the Syria hamster generation that is subjected to 12 type adenovirus infections, can express adenovirus Ela protein.Ela protein activates the GBMT promotor, thereby makes the gene under its control begin to express.2-3 separates the cell clone of anti-MTX after week, and it consumes substratum with the Western hybridization analysis.Identified and severally can in substratum, secrete a kind of clone that can play immunoreactive polypeptide with anti-pphK2 antiserum(antisera).One of them clone (AV12-pGThK2 ^V217#2) selectedly come out to do further evaluation and protein purification.

For purifying hK2 polypeptide, collect AV12-pGThK2 ^V217The serum-free that No. 2 clones of cell cultivated after 7 days consumes substratum, concentrates, and carries out anion-exchange chromatography (Fig. 4 A).Define the active peak of hK2 through elisa assay and near 0.2M NaCl gradient, elute (dotted line).This result of protein band of appearance one treaty 34KD conforms among the SDS/PAGE of ELISA test and same fraction.

Collect the positive fraction of hK2 that anion-exchange column gets off, carry out hydrophobic interaction chromatography (HIC) (Fig. 4 B).A ₂₈₀Major portion do not rest in the HIC post.Main peak rests among the HIC, and this peak eluted at 22 minutes, and has shown the highest activity (dotted line among Fig. 4 C) in the ELISA test.SDS/PAGE also can see the main protein band of a treaty 34KD.Through SEC 22 minutes elution peaks that get off are separated discovery from HIC, the a-protein of general 80-90% ₂₈₀In the time of 19.4 minutes, eluted retention time conform to the about protein of 34KD (Fig. 4 C).Only another protein peak of SEC in the time of 16.7 minutes by wash-out, corresponding to the protein of observed about 70KD in the former purification step.

Be further to determine the protein of purifying, this protein of about 2.5 μ g is added on the automatic N end analyser, obtain following sequence results: Val-Pro-Leu-Ile-Gln-Ser-Arg-Ile-Val-Gly-Gly-Trp-Glu-.The amino acid curve that order discharges in the Edman degraded is not seen tangible competition sequence.Analogized by PSA and to draw, this protein is phK2 ^V217, because the sequence of known ripe PSA (by separating in the seminal fluid) begins to be Ile-Val-Gly-, and pPSA and phK2 are considered at N-terminal 7 extra amino acid (Fig. 2) are arranged.To this proteinic amino acid analysis result and phK2 ^V217The sequence unanimity of inferring.Separate this phK2 polypeptide and purifying and obtain milligram level sample.Embodiment 3phK2 ^V217Evaluation and hK2 ^V217Generation

In order to measure the effect of purification scheme among the embodiment 2, with the phK2 of 1.5 μ g purifying ^V217Carry out the SDS/PAGE electrophoresis under the condition of beta-mercaptoethanol (BME) having or do not have, and dye treatment gel, as a result phK2 in the show sample with silver ^V217Purity be about 95% (Fig. 5), when showing no BME simultaneously, phK2 ^V217With the swimming of the 30KD left and right sides, and when BME is arranged, with the swimming of the 34KD left and right sides.The observations consistent (Fig. 5) of this situation and the PSA of purifying from seminal fluid.

The hK2 aminoacid sequence of inferring from the cDNA sequence shows, locates to exist a potential N-glycosylation site at 78 residues (N-M-S).For determining the whether glycosylation of this site, with phK2 ^V217Carry out the SDS/PAGE electrophoresis, transfer on the nitrocellulose filter,, add anti-DIG antibody response subsequently with horseradish peroxidase institute mark with the reaction of digoxigenin (DIG) link coupled lectin.

In Fig. 6 (1 swimming lane), there is α-mannose residue two non-replacements or that 2-0-replaces in the dyeing prompting protein of concanavalin A matter A (ConA) to 2 μ g phK2,2 swimming lanes are the painted positive control glycoprotein of ConA-monoclonal antibody ZCE025, and it is the N connection oligosaccharides of core that heavy chain of this monoclonal antibody (50KD) and light chain (25KD) all have with the seminose.3 swimming lanes show that non-glycosylated protein (BSA) does not react with the ConA lectin.PhK2 ^V217Also with RCA (Galbl-4GlcNAc specificity, and AAA (α (1-6) connection Fucose specificity) reacts.This lectin reaction formation conforms to the existence of compound N connection oligosaccharides.PhK2 ^V217In oligosaccharides also contain sialic acid because SNA (sialic acid α (2-6) is connected in semi-lactosi) and MAA (sialic acid α (2-6) is connected in semi-lactosi) all can and phK2 ^V217Reaction.

The prosoma sequence of hK2 is VPLIQSR, and the arginine carboxyl terminal enzyme in this precursor sequence is cut and phK2 can be changed into hK2.The gentle tryptic digestion of design makes its phK2 at purifying ^V217This site is with peptide bond hydrolysis.With phK2 ^V217With 1% trypsinase incubation, monitor this conversion process (Fig. 7) with HIC-HPLC.This step makes phK2 ^V217Be converted into hK2 fully ^V217To confirm as is hK2 ^V217The peak carry out the N-end sequencing, prove that its sequence is IVGGWE, this sequence is the N terminal sequence of hK2 mature form just.Do not detect other sequence except that above-mentioned sequence, illustrate that tryptic mild hydrolysis does not produce the non-specific cutting of any obvious level.Find that through the sample of trypsin treatment mobility has very little but can debate other increase, conforms to there being 826 dalton's (preceding peptide molecular weight) very little minimizing on the molecular weight in SDS/PAGE.The preparation of embodiment 4hK2 specific antibody

With phK2 ^V217And hK2 ^V217Make the original monoclonal antibody that produces at hK2 of immunity.With to hK2 ^V217Or phK2 ^V217Hyperergy and be the standard screening hybridoma to the minimal reaction of PSA, the monoclonal antibody representative that obtains from hybridoma is as shown in table 2.With phK2 ^V217Immunity produces HK1G586.1 monoclonal antibody and HK1G464.3 monoclonal antibody.HK1G586.1 is that hK2 is special, because it can discern phK2 ^V217And hK2 ^V217, but nonrecognition PSA.On the other hand, HK1G464 is that phK2 is special, because it only discerns phK2 ^V217, and nonrecognition hK2 ^V217Or PSA.

Table 2 is at hK2 ^V217And phK2 ^V217The specificity of the various monoclonal antibodies that produce

A. at phK2 ^V217The monoclonal antibody that produces

Monoclonal antibody	PSA	?hK2 ^v217	phK2 ^v217
Monoclonal antibody	PSA	?hK2 ^v217	phK2 ^v217	Irrelevant antibody	0.245	?0.162	?0.125
Positive control	2.242±0.06	?9.196	?8.91±0.02	Irrelevant antibody	0.245	?0.162	?0.125
Positive control	2.242±0.06	?9.196	?8.91±0.02	?HK1G586.1 (10μg/ml)	0.150±0.004	?11.154±0.18	?10.146±0.87
HK1G464.3 (ascites 1: 2000)	0.143±0.03	?0.245±0.02	?6.644±0.17	?HK1G586.1 (10μg/ml)	0.150±0.004	?11.154±0.18	?10.146±0.87

B. at hK2 ^V217The monoclonal antibody that produces

The test monoclonal antibody	PSA	?hK2 ^v217	?phK2 ^v217
The test monoclonal antibody	PSA	?hK2 ^v217	?phK2 ^v217	Irrelevant antibody	0.157±0.18	?0.132±0.01	?0.153±0.01
Positive control	2.768±0.08	?8.342±1.3	?9.673±0.99	Irrelevant antibody	0.157±0.18	?0.132±0.01	?0.153±0.01
Positive control	2.768±0.08	?8.342±1.3	?9.673±0.99	Substratum (negative control) only	0.129±0.02	?0.240±0.02	?0.247±0.01
?HK1H247	0.157±0.01	?9.34±0.7	?0.179±0.004	Substratum (negative control) only	0.129±0.02	?0.240±0.02	?0.247±0.01

With hK2 ^V217Immunity produces monoclonal antibody HK1H247.This antibody is that hK2 is special, because it only discerns hK2 ^V217And nonrecognition phK2 ^V217Or PSA.These results show phK2 ^V217And hK2 ^V217Be to produce the effective immunogen that different hK2 forms is had specific monoclonal antibody.

Hybridize with Western and to analyze HK1G586 and whether discern hK2 (Fig. 8) in the seminal fluid.The about 22KD of this monoclonal antibody identification, the hK2 immune response band at 33KD and 85KD place.Nearest Deperthes etc., biological chemistry and biophysics journal, 1245,311 (1995) have also reported hK2 immune response pattern similar in seminal fluid.This result shows at hK2 ^V217Monoclonal anti physical efficiency identification seminal fluid in natural hK2.At hK2 ^V217Or phK2 ^V217All antibody that produce are all discerned the corresponding form of hK2, and this shows that hK2 and phK2 are similar to hK2 respectively on immunology ^V217Or phK2 ^V217(stating as follows).

In order to prepare other anti-hK2 antibody, each member's of human kallikrein gene primary sequence is compared, and with computer-aided analysis antigenicity and hydrophobicity.According to comparative result, several hK2 oligopeptides sequences have been selected.The hK2 peptide that is selected corresponds respectively to the 8-26 position (SEQ ID NO:19) of ripe hK2 amino-acid residue, 15-26 position (SEQ ID NO:26), 41-56 position (SEQ ID NO:20), 43-66 position (SEQ ID NO:24), 153-167 position (SEQID NO:21), 17-71 (SEQ ID NO:22) and 210-235 (SEQ ID NO:25).Synthetic peptide corresponding to the 17-71 amino acids is discerned the possibility of the antibody of natural hK2 form to increase generation.Synthetic and in Mayo Clinic/Foundation protein central laboratory with all peptides of HPLC purifying.With peptide respectively with keyhole relative hemocyanin (KLH) and BSA coupling with as immunogen and analytical reagent.(sheep is SEQ ID NO:20 and 21 to generate polyclonal antibody with KLH-hK2 immunity sheep, goat and mouse, goat is SEQ ID NO:19,20,21,24,25,26) and monoclonal antibody (mouse is SEQ ID NO:19,20,21,24,25 and 26).17-71 peptide (SEQ ID NO:22) oxidation to produce intramolecular disulfide bond between its 26 and 42 halfcystines, is used for immune goat and mouse, to prepare polyclonal antibody and monoclonal antibody respectively.

At first use the hK2 41-56 antibody of hK2 41-56 peptide affinity chromatography column purification, be used for the Western hybridization analysis then from sheep.This antibody capable identification reorganization hK2.HK2 41-56 antibody can be eliminated by the hK2 41-56 peptide of excessive adding the detection of hK2, but can not be eliminated by the PSA41-56 peptide.And the monoclonal antibody of anti-hK2 41-56 peptide in Western analyzes to hK2 protein high special.

The hK2 of antiserum(antisera) (sheep) the identification reorganization of anti-hK2 153-167 peptide.These results show, react with 2 different epi-positions of hK2 polypeptide at the antibody of peptide 41-56 and peptide 153-167.

The antiserum(antisera) of anti-hK2 210-235 amino acids residue has showed the highest immunoreactivity.

HK2 17-71 peptide and PSA corresponding zone homology are 69%, discern reorganization hK2 albumen but nonrecognition PSA at the goat antiserum(antisera) of hK2 17-71 peptide.

The proteinic rabbit anti-serum of reorganization hK2 that directed toward bacteria is expressed can be discerned PSA and hK2, it detects a pair of protein band in the enrichment medium of the LNCaP cell of handling with male sex hormone, in the substratum of the LNCaP cell of handling without male sex hormone, then do not detect immunoreactive protein matter, illustrate that these immunoreactive protein matter are by the male sex hormone inductive.And a top band is the PSA related protein in the LNCaP substratum, because PSA specific antisera (the anti-PSA antiserum(antisera) of rabbit, the reorganization PSA that directed toward bacteria is expressed produces) mainly detects this band.Because have the mouse monoclonal antibody (HK1A523) of monospecific anti-hK2 41-56 peptide to discern a following band in the LNCaP substratum to hK2, this illustrates that it is the hK2 related protein.By this result has also been determined in the sequential analysis of each protein N-end amino acid in this two band.

The histochemical stain result (seeing embodiment 10) who paraffin-embedded human prostate tissue is cut into slices with the monoclonal antibody (HK1A523) of hK2 41-56 peptide shows that as PSA, hK2 results from the epithelial cell, and does not produce in stroma cell.And immunostaining is special to hK2 albumen in the prostata tissue, because hK2 detects all negative in other tissue.The expression of embodiment 5hK2 in mammalian cell

In order in mammalian cell, to express wild-type hK2 (hK2), with pGThK2 (Fig. 3) transfection in the AV12 cell.With HK1D106.4 (a kind of) at the hK2 monoclonal antibody specific that produces corresponding to the polypeptide of hK2 17-71 amino acids residue, by the Western Analysis and Identification clones of several expression hK2 polypeptide.Higher hK2 expression level is selected comes out to be further analyzed because of it for No. 27, AV12-hK2 clone (AV1-hK2).The cell and the HK1D106.4 of transfection empty carrier (pGTD) do not have reactivity.

Carry out the ELISA test shows with the HK1D106.4 monoclonal antibody, having concentration in 7 days AV12-hK2 serum-free of cultivation consumption substratum is the hK2 polypeptide of 0.5-1 μ g/ml.Utilize with from AV12-hK2 ^V217Middle purifying phK2 ^V217Same procedure, cultivate purifying hK2 polypeptide 7 days the consumption substratum from AV2-hK2.The purifying hK2 polypeptide productive rate that obtains is lower, and polypeptide is to the purge process instability.

To carry out the SDS/PAGE electrophoresis through the partially purified hK2 polypeptide of aforesaid method, electricity changes film and carries out the N-terminal amino acid sequencing.The N-terminal sequence of hK2 polypeptide is IVGGWECEK in 7 days the consumption substratum of analysis revealed AV12-hK2 cultivation.Do not see tangible competition sequence by the amino acid collection of illustrative plates that the Edman degradation step discharges in proper order.Learn relatively that with PSA this sequence is corresponding to sophisticated hK2 (hK2).Proteinic amino acid analysis also shows with hK2 and conforms to.

This discovery shows, at AV12-phK2 ^V217The serum-free of cultivating 7 days consumes in the substratum, phK2 ^V217Be major ingredient, and consume in the substratum that hK2 is a main component at the serum-free that AV12-hK2 cultivated 7 days.In order to detect the existence form of hK2 in 1 day the serum free medium of AV12-hK2 cultivation, it is carried out affinity chromatography with partial purification with the HK1G586.1 monoclonal antibody.Carry out the N-terminal analysis after the protein transduction of 34KD moved on to pvdf membrane, obtaining sequence is VPLIQSRIVGG.Do not see tangible competition sequence by the amino acid collection of illustrative plates that the Edman degradation step discharges in proper order.Learn relatively that with PSA this sequence is corresponding to phK2.This announcement is at AV12-hK2 and AV12-hK2 ^V217In the cell, the hK2 polypeptide all is by excretory with precursor forms.Yet phK2 ^V217Stable, do not change into hK2 ^V217, and the phK2 instability changes into hK2 in the extracellular easily.The biosynthesizing of embodiment 6hK2

Be the further biosynthesizing of research hK2 in mammalian cell, the serum-free of collecting No. 27 clones of AV12-hK2 cell every day consumes substratum, and continuous 8 days, carry out the SDS/PAGE electrophoresis after concentrating, carry out time series analysis.Protein transduction is moved on on the nitrocellulose filter and survey (Fig. 9) with HK1D106.4 or HK1G464.3 monoclonal antibody.As shown in Figure 9, HK1D106.4 identification phK2 and hK2 two peptide species, and HK1G464.3 only discerns phK2, because its epi-position is positioned at hK2's-the 7-+7 zone.When detecting with the HK1D1.064 monoclonal antibody, being expressed in the 3rd day and reaching the highest of hK2 polypeptide (about 34KD) enters stationary phase subsequently.Cultivate after 4 days, can detect the immune response band of two other with about 70KD and about 90KD migration.

On the other hand, when surveying, can detect the hK2 level during by the 4th day and reduce gradually with same sample commentaries on classics film and with HK1G464.3.By the 8th day, have only very low-level hK2 to be present in and consume in the substratum.This result shows that the AV12-hK2 cell is secreted into phK2 in the substratum, and gradates outside born of the same parents and be hK2.Curiously, when surveying, do not find this two band of 70KD and 90KD, the homotype oligomer of these bands or hK2 be described with HK1G464.3, or with another agnoprotein matter covalency compound hK2.Though do not determine the identity of these bands at present as yet, they can be used as the existence sign that consumes hK2 in the substratum.Among Fig. 9, with the phK2 of purifying ^V217And hK2 ^V217Albumen is done contrast.

Be research hK2 ^V217Biosynthesizing in the AV12 cell is to AV12-hK2 ^V217No. 2 clones of cell have done a similar time study.As shown in figure 10, survey hK2 with the HK1D106.4 monoclonal antibody ^V217Polypeptide expression reached the highest at the 3rd day, no big variation after the 4th day.Obtain similar result (Figure 10) when surveying this trace as probe with the HK1G464.3 monoclonal antibody.These results show, AV12-pGThK2 ^V217No. 2 clones of cell expressed phK2 since the 1st day ^V217, continuous expression in subsequently 8 days at least, this protein is not converted into mature form.These results are different with the result of phK2, if kept 8 days in substratum, phK2 can change into hK2, and this has just illustrated phK2 ^V217In 37 ℃ of substratum, be stable in 8 days.

For whether research extracellular phK2 is relevant with No. 27 clones' of AV12-hK2 cell vigor in the culture to the conversion of hK2, No. 27 clone cells are counted with trypan blue exclusion.With HK1D106.4 and two kinds of monoclonal antibodies of HK1G464.3 ELISA being carried out in the expression that consumes hK2 in the substratum detects.As shown in figure 11, the quantity of survivaling cell is the highest in the time of the 3rd day in the culture, reaches 3,800 ten thousand, reduces gradually subsequently.By the 8th day, survivaling cell reduced to 1,000 ten thousand.The expression of phK2 (detecting with HK1G464.3) also reached the highest at the 3rd day, and reduced gradually subsequently.

On the other hand, the expression of hK2 (HK1D106.4 detection) reached peak value at the 3rd day, entered stationary phase subsequently.This result shows AV12-hK2 emiocytosis phK2, and during by the 4th day, a part of phK2 is gradually transformed into hK2 outside born of the same parents.In addition, the result shows that phK2 is obviously relevant with the reduction of cell viability to the conversion of hK2, shows that the extracellular protease that is discharged by dead cell may be one of factor of this conversion.The expression of hK2 was the highest when cell had vigor most.The minimizing of hK2 is parallel to the reduction of cell viability, shows hK2 by these emiocytosis, rather than just discharges after these necrocytosiss and cracking.Simultaneously, the rising of hK2 is corresponding with the reduction of phK2, and the development along with the time is described, the precursor forms of hK2 is converted into mature form automatically.

For detecting the biosynthesizing of hK2 in prostate cancer cell, hK2 is expressed in DU145 and PC3 clone.In pLNCX and pLNSX plasmid (Miller and Rosman, biotechnology, 7,980 (1989)), make it respectively under the control of CMV and SV40 promotor the dna clone of coding pphK2, obtain pLNC-hK2 and pLNS-hK2 plasmid respectively.These two kinds of plasmids are distinguished transfection in PC3 and DU145 cell, and in containing the substratum of G418, screen.Screen the clone (PC3-hK2 and DU145-hK2) of high level expression hK2 with the method for ELISA and Western hybridization.

In order to estimate hK2 and the phK2 expression level in substratum, utilize monoclonal antibody HK1D106.4 (hK2 is special) and HK1G464.3 (phK2 is special), the serum free medium of PC3-hK2 and DU145-hK2 cell is carried out Western hybridization (Figure 12).The result shows that phK2 is present in the consumption substratum of DU145-hK2 and PC3-hK2, shows that hK2 is with phK2 form excretory in the prostate cancer cell, and change into mature form outside born of the same parents.The result who has obtained by the AV12 cell before this discovery has confirmed.Even after cultivating 7 days, phK2 is a dominant component still in the consumption substratum of PC3-hK2 cell, and since the 1st day, and hK2 is the dominant component in the serum free medium of DU145-hK2 cell.This may be to have abundant extracellular protease in the substratum because DU145 consumes.

Whether only be confined to 1 clone in order to detect above result, detected the AV12-hK2 clone of other 3 independent separate and the AV12-hK2 of other 4 independent separate ^V217HK2 polypeptide expression among the clone.Collected these clones' serum free medium at the 7th day, utilizing HK1D106.4 (HK2 is special) and HK1G464 (phK2 is special) monoclonal antibody is probe, Western is carried out in the expression of hK2 analyze (Figure 13 and 14).In all AV12-hK2 clones, the HK1D106.4 monoclonal antibody not only detects main 34KD band (hK2), also detects 70KD and 90KD band (Figure 13) that indication hK2 exists.HK1G464.3 detects very low-level phK2 (Figure 14) in all AV12-hK2 clones.This result shows, mainly has hK2 in the consumption substratum of all AV12-hK2 cell clones, has verified the biosynthesizing mechanism of setting up in No. 27 clones of AV12-hK2 cell.To AV12-hK2 ^V217The clone is cooked same analysis (Figure 14), and the result shows in these consumption substratum of cloning the 7th day only phK2 ^V217Existence, verified that we are at AV12-hK2 ^V217Discovery among the clone.

In sum, above result shows that hK2 expresses with precursor forms in mammalian cell, and quilt ignorant enzymatic conversion still is a mature form outside born of the same parents.These results show that also phK2 may be present in the biological liquid, therefore can be as the useful diagnosis index of pCa and BPH.Embodiment 7hK2 and hK2 ^V217Enzymic activity and specificity

A small amount of hK2 is purified to the purity that is enough to be used for analyzing its enzymic activity and substrate specificity, by measuring hK2 detects hK2 to the adding lustre to property amidohydrolase activity of the p-nitrophenyl sulfonamide derivatives of peptide general activity (table 3).With A ₄₀₅The proteolysis matter digestion of these substrates of absorption measurement in the p-Nitroaniline that discharges.Measure the chymotrypsin-like protease activities with substrate methoxyl group succinyl--Arg-Pro-tyrosine-p-Nitroaniline (MeO-Suc-R-P-Y-pNA), the cleavage site of this enzyme is at phenylalanine.This substrate was used to detect the activity (Christensson etc., european journal of biological chemistry, 194,755 (1990)) of PSA in the past.Substrate H-D-proline(Pro)-phenylalanine-arginine-p-Nitroaniline (P-F-R-pNA) is that trypsin-like proteolytic enzyme is special, and this enzyme cleavage site is at arginine (R).

HK2 compares hK2 to the gross activity of P-F-R-pNA substrate ^V217High 10 times, but both all do not show the hydrolysis ability to chymotrypsin protein enzyme substrates MeO-Suc-R-P-Y-pNA.Test the comparable substrate that other contains trypsin-like cleavage site (Methionin, arginine), find that the speed of hK2 hydrolysis P-F-R-pNA substrate is the highest, these discoveries show that hK2 has the trypsin-like activity.

Table 3hK2, hK2 ^V217, PSA and trypsinase is to the amidohydrolase activity of chromogenic substrate

Proteolytic enzyme	?MeO-Suc-R-P-Y-pNA ?xmol/min/μg/ml	?P-F-R-pNA ?xmol/min/μg/ml
Proteolytic enzyme	?MeO-Suc-R-P-Y-pNA ?xmol/min/μg/ml	?P-F-R-pNA ?xmol/min/μg/ml	?hK2 ^v217	?0	?8.7pmol
?hK2	?0	?4.1nmol	?hK2 ^v217	?0	?8.7pmol
?hK2	?0	?4.1nmol	?PSA	?13.3pmol	?2.2pmol
Trypsinase	?3.8pmol	?25.5nmol	?PSA	?13.3pmol	?2.2pmol

Use peptide substrates and the peptide bond of N-terminal amino acid sequence analysis instrument to determine to be hydrolyzed, thereby to hK2 and hK2 ^V217Specificity carry out detailed analysis.Figure 15 has shown the amidohydrolase activity to peptide C ALPEKPAVYTKVVHYRKWIKDTIAAN, and this polypeptide contains the potential point of contact of trypsinase and two kinds of enzymes of Quimotrase.HK2 ^V217All can cut at trypsinase site (R-K) and Quimotrase site (Y-R), and its cutting in the trypsinase site is 2: 1 with its cutting ratio in the Quimotrase site.As the contrast of these experiments, also with phK2 ^V217With the common incubation of this peptide, do not find amidohydrolase activity.HK2 has showed and hK2 this peptide substrates ^V217Different specificitys.Do not find that hK2 has the chymotrypsin-like specificity to this substrate, its activity is all at trypsin-like site (R-K).Other lysine residue all is not hydrolyzed in this polypeptide, and the specificity that hK2 is described is only in arginine residues.

In contrast, trypsinase also is used for research to this substrate, all Methionins (K) and arginine (R) site of trypsinase cutting except that K-P key (known is not tryptic suitable cleavage site).To the R-K site of 210-236 substrate (Figure 16, No. 1 peptide), approximately than hK2 fast 4 times of tryptic cutting speed in feet per minutes compare hK2 ^V217Fast about 4000 times.Trypsinase does not cut the chymotrypsin-like site.PSA mainly cuts the Y-R key.Find that also PSA has the more weak trypsin-like activity (Figure 15) to the R-K key, this found with former that PSA had the active result of more weak trypsin-like to conform to chromogenic substrate (table 2).

Also with other several peptide substrates and hK2 and the common incubation of PSA (Figure 16).In all test peptides, hK2 only has specificity to some arginine, and PSA mainly has specificity to some tyrosine (Y), phenylalanine (F) and leucine (L) residue.Has only No. 1 peptide among Figure 16 by hK2 ^V217Cutting has detailed description in the color atlas of Figure 15.Embodiment 8hK2 is to phK2 ^V217Activation

Among Figure 16 the sequence of No. 3 peptides corresponding to phK2-amino-acid residue of 7-+7 position.This district comprises propetide VPLIQSR, has found that this is phK2 ^V217The N-terminal leading peptide of polypeptide.As mentioned above, hK2 can cut this peptide and discharge pre-peptide region, and hK2 ^V217Then can not.In order to describe this pre-peptide region whether hK2 can cut natural substrate, to it with phK2 ^V217Change into hK2 ^V217Ability monitor.With phK2 ^V217With the common incubation of 1%hK2, with HIC-HPLC method monitoring conversion process (Figure 17 A).The result shows that hK2 can be with phK2 ^V217Be converted into hK2 ^V217, but its conversion rate than trypsinase slow nearly 30 times.Work as phK2 ^V217And 40%hK2 ^V217During common incubation, even through 6 hours, the ratio of two kinds of hK2 forms does not change (Figure 17 B) yet.Before having confirmed, this result, also shows, to have only hK2 can cut the pre-peptide region of hK2 even for natural substrate to the observation of this peptide substrates, and hK2 ^V217Then can not.

These results comprehensively show, even prolong the incubation time, phK2 ^V217And hK2 ^V217Also be stable.With hK2 ^V217Compare, hK2 has the specificity of higher proteolytic activity and Geng Gao, particularly has the specificity to the hK2 precursor forms, among Figure 15 among the activity of precursor peptide and Figure 17 to phK2 ^V217Activity all confirmed this specific specificity.

These results confirm hK2 and hK2 ^V217There were significant differences aspect enzymic activity, and have low-yield when helping explain from substratum purifying hK2 (with phK2 ^V217Compare).As seeing in other active protease, highly purified hK2 preparation may be because self hydrolysis and instability.These results further point out, and are except immunity test, also available to this proteinic level in the enzymic activity monitoring body fluid of hK2 specific substrate.Embodiment 9 and hK2 form inhibitor complexes

PSA energy and α ₂Macroglobulin (MG), serpin chymotrypsin inhibitor (ACT) forms mixture.In order to study the formation of hK2 mixture, with common a series of proteolytic enzyme (ACT, α in itself and the human plasma ₂-antiplasmin, anticoagulin III and α ₁-antitrypsin (Trayis and Salvesen, biological chemistry year looks back 52,655 (1983)) is incubation together, and mixture is carried out Western hybridization analysis (Figure 18).Under reductive condition, any covalent complex that hK2 and these serpins form all obtains the band of about 80-100KD.

ACT and α ₂-antiplasmin and hK2 form significant mixture (Figure 18,1 swimming lane and 2 swimming lanes), anticoagulin III (3 swimming lane) and α ₁-antitrypsin (α ₁Proteinase inhibitor, 4 swimming lanes) do not form detectable mixture with hK2.A kind of main component MG of plasma proteins can be rapidly and hK2 compound (5 swimming lane), and this mixture corresponding molecular weight is about 200KD and 120KD, also can form such mixture (Figure 18,8 swimming lanes are stated as follows) when PSA and the purified common incubation of MG.Absorbingly be, though this albumen mass-energy of alpha1-antitrypsin suppresses many trypsin-like proteolytic enzyme (Loebermann etc., molecular biology magazine, 177,531 (1984); Carrell and Trayis, TIBS, 10,20, (1985)), but hK2 can not form mixture with it.

HK2 and α ₂Antiplasmin forms the mixture no wonder, because α ₂Antiplasmin contains arginine residues (Hunt and Dayhoff, biological chemistry and biophysical studies communication, 95,864 (1980) in its inhibitor activity site; Chardra etc., biological chemistry, 22,5055 (1983); Potempa etc., science, 241,699 (1985); Shieh etc., journal of biological chemistry, 264,13420 (1989); Mast etc., biological chemistry, 30,1723 (1991)).It but is beyond the consideration that yet hK2 and ACT form mixture, because ACT has a leucine in its inhibitor activity site.Though Figure 16 and table 2 show PSA and hK2 and have diverse proteolysis specificity, their similaritys have structurally obviously influenced the character that they and common inhibitor form mixture.

Western hybridization (Figure 18) shows that the hK2 that is added in human women's serum can rapid and MG formation mixture.1 swimming lane and 3 swimming lanes are contrasts that hK2 and serum are only arranged respectively.2 swimming lanes are the hK2 with the common incubation of ACT, demonstrate hK2-ACT mixture and the remaining hK2 of 90KD.4 swimming lanes and 5 swimming lanes are respectively the hK2 that adds in the serum 15 minutes and 1 hour.6 swimming lanes are and purified 4 hours hK2 of MG incubation.7 swimming lanes are the PSA that added in the serum 15 minutes, and 8 swimming lanes are and purified 4 hours PSA of MG incubation.

The result shows, in experiment in vitro, when being added to hK2 or PSA in the human serum, MG is a composition main and hK2 or PSA formation mixture.Because known in prostatosis patient's serum PSA and ACT form mixture, people be sure of that the hK2 that exists in the serum also can form the ACT mixture of certain level.Discuss

Still do not know at present PSA or hK2 protein processing and mechanism of secretion in vivo.Result of the present invention shows that AV12-hK2, DU145-hK2 and PC3-hK2 cell all can be secreted phK2, illustrates under the normal circumstances, and hK2 is that this precursor peptide is cut outside born of the same parents with phK2 form excretory.This prompting phK2 is present in the biological liquid, therefore can be used for diagnosing the level of signification of pCa or BPH.

Mutant hK2 (hK2 ^V217) and wild-type hK2 can both from the AV12 cell, obtain by purifying.As what find in other proteolytic enzyme, possibility is owing to himself catalytic ability, and hK2 is very unstable to the purge process of using, and makes the hK2 of purifying q.s or phK2 very difficult to be used as immunogen and standard substance.On the contrary, phK2 ^V217Highly stable, can be generated also very stable hK2 by tryptic digestion ^V217The phK2 of purifying ^V217And hK2 ^V217Provide immunogen for producing hK2 and phK2 monoclonal antibody specific.The immunoreactivity of 586 pairs of prostata tissues of embodiment 10 monoclonal antibodies

With HK1523 normal prostate tissue is carried out immunohistochemical staining and find that dyeing appears at epithelial cell but not stroma cell.And the expression of hK2 is a prostate-specific, because other tissue, can not be painted as kidney and pancreas.In order to determine whether hK2 expresses in prostata tissue, if, whether relevant with prostate cancer, obtaining 264 prostate glands excises sample entirely and compares analysis, wherein 257 sample sources are in not subject patient (Figure 20), the hang oneself patient (Figure 21) of the hungry treatment of male sex hormone of 7 samples.Analyzing the kytoplasm of optimum epithelial cell district in each sample, the interior sarcoma district (PIN) of height prostatic epithelium and adenocarcinoma cell district hK2 expresses.

Prostata tissue is weighed three-dimensional measurement and record.Top and bottom respectively cut behind the 4-5mm with the continuous stripping and slicing of 3mm.The residue prostate gland to the seminal vesicle tip, uses cutter with 4-5mm spacing continuous stripping and slicing perpendicular to the body of gland major axis from the body of gland top.Make transverse section, use hematoxylin-eosin staining.The cancer patient of the full excision of each prostate gland selects a section that had not only comprised cancerous tissue but also comprised benign tissue, and the technology that adopts this area to understand thoroughly is fixed in the 10% neutral formalin damping fluid and uses paraffin embedding.

Tissue slice on the slide glass is immersed earlier dimethylbenzene to be immersed in 95% ethanol again and dewaxes.In order to seal endogenous peroxidase activity, the incubation 10 minutes of will cutting into slices in methyl alcohol/hydrogen peroxide, tap water flushing then.Subsequently section is placed the 10mM citrate buffer of pH6.0, steam treatment 30 minutes.The section cooling is after 5 minutes, and cold running water flowing water washes.Section combines to seal nonspecific proteins matter with 5% lowlenthal serum incubation 10 minutes, and gentleness is dried section then.

One anti-(hK1G586 of 0.5 μ g/ml or PSM773) is added in the section, and room temperature left standstill 30 minutes, then with tap water flushing section, will cut into slices subsequently and biotin labeled rabbit anti-mouse antibody incubation 1 hour, and tap water washes.To cut into slices and peroxidase link coupled streptavidin (1: 500) incubation 30 minutes tap water flushing then.Subsequently, will cut into slices and 3-amino-9-ethyl carbazone (ABC) chromogenic agent solution incubation 15 minutes tap water flushing again.To cut into slices then and use no mercury haematoxylin redyeing 1 minute, flowing water flushing 5 minutes.It is fixing to cut into slices with water-soluble bonding die agent (glycogelatin).To optimum epithelial cell, height PIN and gland cancer counting staining cell, wherein classify from 0% to 100% by 10% system.

The dyeing of optimum atrophy body of gland is minimum, particularly areas of inflammation does not almost have immunoreactivity, the hyperplasia acinus or the optimum acinus that do not have obvious atrophy have moderate to arrive the intensive immunoreactivity, are particulate state usually in the secretion chamber cellular layer above nuclear and distribute, and often extend to the surface, chamber.The urothelial of urethra and viva montanum is not painted, although following body of gland usually shows immunoreactivity.The basilar cell is often negative.

The sample that height PIN is arranged is in whole kytoplasm, in most cases demonstrate the intensive immunoreactivity in the vertical vesicle of kytoplasm.Immunoreactivity there is no significant difference in dissimilar PIN, and just in the Cribiform type, the intensity of middle body is more shallow than the edge section usually.

In fact the reaction of intensive kytoplasm is all arranged in all cancer samples.The cell that contains more kytoplasm bubble is painted more shallow, comprises finger ring shape cell and Saliva Orthana zone, and other kytoplasm is dyeing strongly all.Intensive dyeing appears in the gland cancer of highest ranking (Gleason level Four), and it all shows immunoreactivity in all cases.Have the focus of Cribiform cancer to be similar to CribiformPIN, similarity is that edge dyeing is strong than the center.The edge in the territory or the forward position of cancer development the intensive immunoreactivity is often arranged.

In 7 samples through male sex hormone starvation cures treatment patients, in most of optimum atrophic acinuses immunological unresponsiveness or a little less than, though PIN and gland cancer show the dyeing of intensive kytoplasm sometimes.

With monoclonal antibody HK1G586 optimum epithelium, height PIN and gland cancer are dyeed, its cell dyeing counting is summarized in the table 4.To its pair analysis, promptly optimum and PIN pairing, optimum and cancer pairing, PIN and cancer are matched and can be disclosed significant difference (P＜0.001, Spearman Rank Correlation) between all kinds of.

The immunoreactivity of table 4 and hk1G586

	Mean value	Standard deviation (%)
	Mean value	Standard deviation (%)	Optimum epithelium	44.3％	10-90
Height PIN	69.1％	20-100	Optimum epithelium	44.3％	10-90
Height PIN	69.1％	20-100	Gland cancer (untreated)	80.0％	20-100

Like this, the increase that kytoplasm hK2 expresses in the prostata tissue is relevant with hyperplasia of prostate and prostate cancer.Though because sample is less, it is not too remarkable statistically to derive from the patient's data for the treatment of through the male sex hormone starvation cure, not compare with treating patient, the expression of these patients hK2 in optimum epithelium, height PIN and gland cancer all reduces to some extent.So the rising that hK2 expresses in the prostate gland is a New Set of height PIN and prostate cancer.The RT-PCR of hK2 RNA detects in embodiment 11 prostate cancer cells

Because most of prostate cancers are recessive, thus from the biopsy sample (as capsula prostatica, marrow or lymphoglandula) or physiological liquid (as blood, serum or seminal fluid) to detect the cell of expressing hK2 just very effective.This detection method preferably can detect the individual cells of expressing hK2 from the cell of not expressing hK2 in a large number.Preferably, this method can be from comprising at least about 10 ⁴Detecting the individual cells of expressing hK2 in the sample of individual cell, more preferably is from comprising at least 10 ⁶About cell, or more preferably 10 ⁷About detect the individual cells of expressing hK2 in the sample of cell.For a kind of so quick detection method of recording is provided, used the special reverse transcription-polymerase chain reaction (RT-PCR) of hK2 transcript.

A.LNCaP clone

In order to measure the susceptibility that detects hK2 specific transcriptional thing with the RT-PCR method, with the LNCaP clone serial dilution of expressing human PSA and hK2 in the buffy layer cell.The buffy layer cell obtains by separating in normal male or women's the whole blood.In Citrate trianion-D-glucose pipe, collect 5-7ml venous blood, under 4 ℃, with sample centrifugal 15 minutes at 1000xg.Recovered overhead buffy layer cell from cell mass.

Change with per minute 1500,, from sedimentary cell mass, extract RNA then centrifugal 5 minutes of buffy layer cell and LNCaP cell mixture.Adopt sour phenol-chloroform-guanidinium isothiocyanate method isolation of RNA (Chomczynski etc., analytical chemistry, 162,156,1987) with chloroform-T alcohol (4: 1, volume ratio) further extracting RNA is to remove residual protoheme, and protoheme can suppress reverse transcription and polymerase chain reaction.Handle isolating RNA with the DNA enzyme of no RNA enzyme then.

First chain for preparation cDNA, the sample that portion is contained the total RNA of 1 μ g joins in the reverse transcription reaction system, this reverse transcription reaction system contains the special Oligonucleolide primers of 100pmol PSA (5 ' TCATCTCTGTATCC 3 ', SEQ ID NO:13) or the special Oligonucleolide primers (5 ' GAGTAAGCTCTA 3 ' of 100pmol hK2, SEQ ID NO:14) and Moloney murine leukemia virus reverse transcriptase (GIBCO BRL).Final volume is transferred to 25 μ l (50mM Tris-hydrochloric acid, pH8.3,75mM Repone K, 3mM magnesium chloride, 10mM dithiothreitol (DTT), every kind of dNTP of 0.5mM, and 800 Moloney of unit murine leukemia virus reverse transcriptases).To be reflected at 42 ℃ of incubations 15 minutes, 95 ℃ of heating made the hot deactivation of enzyme in 15 seconds then.

Be the amplification PSA first chain cDNA, right with the primer that PSA is special, the first chain cDNA, the 10 μ l that will be caused by the special oligonucleotide of PSA are at PCR (every kind of dNTP of 0.2mM, 0.5 the AmpliTaq of unit polysaccharase, 50mM Repone K, 10mM Tris-hydrochloric acid, pH8.3,1.5mM magnesium chloride, 0.1% (w/v) gelatin) increases in.The pcr amplification of PSA has utilized the PSA-1 (5 ' GATGACTCCAGCCACGACCT 3 ', SEQ ID NO:1 5) of 50pmol and the PSA-2 (5 ' CACAGACACCCCATCCTATC 3 ', SEQ ID NO:16) of 50pmol.For the first chain cDNA of the hK2 that increases, with the special primer of hK2 to the first chain cDNA that will cause by the special oligonucleotide of hK2 at PCR (every kind of DNTP of 0.2mM, 0.5U AmpliTaq polysaccharase, 50mM KCl, 10mMTris-HCl, pH8.3,1.5mM MgCl ₂, 0.1% (w/v) gelatin) in increase.The pcr amplification of hK2 has utilized 50pmol hK2-1 (5 ' GAGGGTTGTGTACAGTCATGGAT3 ', SEQID NO:17) and 50pmol hK2-2 (5 ' ACACACTGAAGACTCCTGGGGCG 3 ', SEQID NO:18).

Used loop parameter is as follows: 35-40 circulation, and each circulation is 94 ℃, 1 minute; 58 ℃ (PSA) or 60 ℃ (hK2), 90 seconds; 72 ℃, 90 seconds.Last circulation be 72 ℃ 10 minutes.Sample electrophoresis in 1.0% sepharose is answered in negate, observes under ultraviolet lamp behind the ethidium bromide staining.The part amplified production is scaled off from gel, and subclone to pCR II carrier (Invitrogen, San Diego, CA) in for order-checking.

The special pcr amplification of PSA goes out the product of a 710bp, and the special pcr amplification of hK2 goes out the product of a 405bp.Dilution analysis is the result show, respectively 10 ⁶With 10 ⁷Can effectively detect expressed PSA of single LNCaP cell or hK2 RNA (Figure 22 A) in the individual white corpuscle.Very surprising is that RT-PCR detects the PSA transcript high 10 times extent of dilution of the hK2 transcript in LNCaP source than the LNCaP source.B. patients with prostate cancer

With the blood sample of RT-PCR analysis from 6 patients with prostate cancer and 2 normal males.Press preceding method, from 8 male sex, obtain the buffy layer cell and extract RNA, carry out the RT-PCR reaction.In 6 patients with prostate cancer, comprise 1 clinical B phase patients with prostate cancer, 2 patient's (clinical D2 phase) and 3 pathology C phase patients that known generation is shifted.The prostate cancer of pathology A-C phase is the prostate cancer of localized forms, and pathology D1 phase prostate cancer has been diffused into lymphoglandula (nodus lymphoideus transferring rate), and the pathology D2 phase is general prostate cancer (general transfer).About the detailed content of prostate cancer pathological stage, referring to (cancer research, 52,6110 (1992)) such as Moreno, (urology, 43,765 (1994)) such as Deguchi etc. (cancer research 53,5350 (1993)) and Katz.

The result shows 67% patients with prostate cancer expression hK2, and 17% expresses PSA, and 17% had both expressed hK2 also expresses PSA.In the normal control group, but the PSA or the hK2 RNA of nothing detection level.

Like this, the detection of hK2 RNA can be used as an effective marker of prostate cancer micrometastasis early detection.

The present invention is not limited to shown and details that narrated, because in described invention essence of claim of the present invention and scope, can do many modifications and variations.

Sequence table (1) general information (ⅰ) applicant: Mayo Foundation for Medical Education and

Resarch, and Hybritech Incorporated (ⅱ) denomination of invention: the detection method of metastatic prostate cancer (ⅲ) sequence number: 26 (ⅳ) mailing address

(A) address: Schwegman, Lundberg, Woessner and Kluth, P.A.

(B) street: P.O.Box 2938

(C) city: Minneapolis

(D) state: MN

(E) country: the U.S.

(F) postcode: 55402 (ⅴ) computer-reader form

(A) media type: disk

(B) computer: IBM compatible

(C) operating system: DOS

(D) software: this application information of Fast SEQ Version 2.0 (ⅵ)

(A) application number: the unknown

(B) applying date: on November 14th, 1997

(C) classification: (ⅶ) application information formerly

(A) application number: 08/759,354

(B) applying date: on November 14th, 1996

(A) application number: PCT/US96/06167

(B) applying date: on May 2nd, 1996

(A) application number: 08/622,046

(B) applying date: on March 26th, 1996

(A) application number: 08/427,707

(B) submit the date: May 2 nineteen ninety-five (ⅷ) agency/proxy's information

(A) name: Embretson, Janet E

(B) registration number: 39,665

(C) reference/number of documents: 545.005WO1 (ⅸ) telecommunications contact details

(A) phone: 612-359-3260

(B) fax: 612-359-3263

(C) telegram: (2) SEQ ID NO:1 information: (ⅰ) sequence signature:

(A) length: 237 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:1:Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val1 5 10 15Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His

20??????????????????25??????????????????30Pro?Gln?Trp?Val?Leu?Thr?Ala?Ala?His?Cys?Leu?Lys?Lys?Asn?Ser?Gln

35??????????????????40??????????????????45Val?Trp?Leu?Gly?Arg?His?Asn?Leu?Phe?Glu?Pro?Glu?Asp?Thr?Gly?Gln

50??????????????????55??????????????????60Arg?Val?Pro?Val?Ser?His?Ser?Phe?Pro?His?Pro?Leu?Tyr?ASn?Met?Ser65??????????????????70??????????????????75??????????????????80Leu?Leu?Lys?His?Gln?Ser?Leu?Arg?Pro?Asp?Glu?Asp?Ser?Ser?His?Asp

85??????????????????90??????????????????95Leu?Met?Leu?Leu?Arg?Leu?Ser?Glu?Pro?Ala?Lys?Ile?Thr?Asp?Val?Val

100?????????????????105?????????????????110Lys?Val?Leu?Gly?Leu?Pro?Thr?Gln?Glu?Pro?Ala?Leu?Gly?Thr?Thr?Cys

115?????????????????120?????????????????125Tyr?Ala?Ser?Gly?Trp?Gly?Ser?Ile?Glu?Pro?Glu?Glu?Phe?Leu?Arg?Pro

130?????????????????135?????????????????140Arg?Ser?Leu?Gln?Cys?Val?Ser?Leu?His?Leu?Leu?Ser?Asn?Asp?Met?Cys145?????????????????150?????????????????155?????????????????160Ala?Arg?Ala?Tyr?Ser?Glu?Lys?Val?Thr?Glu?Phe?Met?Leu?Cys?Ala?Gly

165?????????????????170?????????????????175Leu?Trp?Thr?Gly?Gly?Lys?Asp?Thr?Cys?Gly?Gly?Asp?Ser?Gly?Gly?Pro

180?????????????????185?????????????????190Leu?Val?Cys?Asn?Gly?Val?Leu?Gln?Gly?Ile?Thr?Ser?Trp?Gly?Pro?Glu

195?????????????????200?????????????????205Pro?Cys?Ala?Leu?Pro?Glu?Lys?Pro?Ala?Val?Tyr?Thr?Lys?Val?Val?His

210 215 220Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro225,230 235 (2) SEQ ID NO:2 information: (ⅰ) sequence signature:

(A) length: 711 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological structure: linearity, (ⅱ) molecule type: cDNA, (ⅹ ⅰ) sequence description: SEQ ID NO:2:ATT GTG GGA GGC TGG GAG TGT GAG AAG CAT TCC CAA CCC TGG CAG GTG 48Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val 15 10 15GCT GTG TAC AGT CAT GGA TGG GCA CAC TGT GGG GGT GTC CTG GTG CAC 96Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His

20??????????????????25??????????????????30CCC?CAG?TGG?GTG?CTC?ACA?GCT?GCC?CAT?TGC?CTA?AAG?AAG?AAT?AGC?CAG????144Pro?Gln?Trp?Val?Leu?Thr?Ala?Ala?His?Cys?Leu?Lys?Lys?Asn?Ser?Gln

35??????????????????40??????????????????45GTC?TGG?CTG?GGT?CGG?CAC?AAC?CTG?TTT?GAG?CCT?GAA?GAC?ACA?GGC?CAG????192Val?Trp?Leu?Gly?Arg?His?Asn?Leu?Phe?Glu?Pro?Glu?Asp?Thr?Gly?Gln

50??????????????????55??????????????????60AGG?GTC?CCT?GTC?AGC?CAC?AGC?TTC?CCA?CAC?CCG?CTC?TAC?AAT?ATG?AGC????240Arg?Val?Pro?Val?Ser?His?Ser?Phe?Pro?His?Pro?Leu?Tyr?Asn?Met?Ser?65??????????????????70??????????????????75??????????????????80CTT?CTG?AAG?CAT?CAA?AGC?CTT?AGA?CCA?GAT?GAA?GAC?TCC?AGC?CAT?GAC????288Leu?Leu?Lys?His?Gln?Ser?Leu?Arg?Pro?Asp?Glu?Asp?Ser?Ser?His?Asp

85??????????????????90??????????????????95CTC?ATG?CTG?CTC?CGC?CTG?TCA?GAG?CCT?GCC?AAG?ATC?ACA?GAT?GTT?GTG????336Leu?Met?Leu?Leu?Arg?Leu?Ser?Glu?Pro?Ala?Lys?Ile?Thr?Asp?Val?Val

100?????????????????105?????????????????110AAG?GTC?CTG?GGC?CTG?CCC?ACC?CAG?GAG?CCA?GCA?CTG?GGG?ACC?ACC?TGC????384Lys?Val?Leu?Gly?Leu?Pro?Thr?Gln?Glu?Pro?Ala?Leu?Gly?Thr?Thr?Cys

115?????????????????120?????????????????125TAC?GCC?TCA?GGC?TGG?GGC?AGC?ATC?GAA?CCA?GAG?GAG?TTC?TTG?CGC?CCC????432Tyr?Ala?Ser?Gly?Trp?Gly?Ser?Ile?Glu?Pro?Glu?Glu?Phe?Leu?Arg?Pro

130?????????????????135?????????????????140AGG?AGT?CTT?CAG?TGT?GTG?AGC?CTC?CAT?CTC?CTG?TCC?AAT?GAC?ATG?TGT????480Arg?Ser?Leu?Gln?Cys?Val?Ser?Leu?His?Leu?Leu?Ser?Asn?Asp?Met?Cys145?????????????????150?????????????????155?????????????????160GCT?AGA?GCT?TAC?TCT?GAG?AAG?GTG?ACA?GAG?TTC?ATG?TTG?TGT?GCT?GGG????528Ala?Arg?Ala?Tyr?Ser?Glu?Lys?Val?Thr?Glu?Phe?Met?Leu?Cys?Ala?Gly

165?????????????????170?????????????????175CTC?TGG?ACA?GGT?GGT?AAA?GAC?ACT?TGT?GGG?GGT?GAT?TCT?GGG?GGT?CCA????576Leu?Trp?Thr?Gly?Gly?Lys?Asp?Thr?Cys?Gly?Gly?Asp?Ser?Gly?Gly?Pro

180?????????????????185?????????????????190CTT?GTC?TGT?AAT?GGT?GTG?CTT?CAA?GGT?ATC?ACA?TCA?TGG?GGC?CCT?GAG????624Leu?Val?Cys?Asn?Gly?Val?Leu?Gln?Gly?Ile?Thr?Ser?Trp?Gly?Pro?Glu

195?????????????????200?????????????????205CCA?TGT?GCC?CTG?CCT?GAA?AAG?CCT?GCT?GTG?TAC?ACC?AAG?GTG?GTG?CAT????672Pro?Cys?Ala?Leu?Pro?Glu?Lys?Pro?Ala?Val?Tyr?Thr?Lys?Val?Val?His

210 215 220TAC CGG AAG TGG ATC AAG GAC ACC ATC GCA GCC AAC CCC 711Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro225,230 235 (2) SEQ ID NO:3 information: (ⅰ) sequence signature:

(A) length: 261 amino acid

(B) type: amino acid

(D) topological framework: linear (ⅱ) molecule type: protein (ⅹ ⅰ) sequence description: SEQ ID NO:3:Met Trp Asp Leu Val Leu Ser Ile Ala Leu Ser Val Gly Cys Thr Gly 15 10 15Ala Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gly Trp Glu Cys Glu

20??????????????????25??????????????????30Lys?His?Ser?Gln?Pro?Trp?Gln?Val?Ala?Val?Tyr?Ser?His?Gly?Trp?Ala

35??????????????????40??????????????????45His?Cys?Gly?Gly?Val?Leu?Val?His?Pro?Gln?Trp?Val?Leu?Thr?Ala?Ala

50??????????????????55??????????????????60His?Cys?Leu?Lys?Lys?Asn?Ser?Gln?Val?Trp?Leu?Gly?Arg?His?Asn?Leu?65??????????????????70??????????????????75??????????????????80Phe?Glu?Pro?Glu?Asp?Thr?Gly?Gln?Arg?Val?Pro?Val?Ser?His?Ser?Phe

85??????????????????90??????????????????95Pro?His?Pro?Leu?Tyr?Asn?Met?Ser?Leu?Leu?Lys?His?Gln?Ser?Leu?Arg

100?????????????????105?????????????????110Pro?Asp?Glu?Asp?Ser?Ser?His?Asp?Leu?Met?Leu?Leu?Arg?Leu?Ser?Glu

115?????????????????120?????????????????125Pro?Ala?Lys?Ile?Thr?Asp?Val?Val?Lys?Val?Leu?Gly?Leu?Pro?Thr?Gln

130?????????????????135?????????????????140Glu?Pro?Ala?Leu?Gly?Thr?Thr?Cys?Tyr?Ala?Ser?Gly?Trp?Gly?Ser?Ile145?????????????????150?????????????????155?????????????????160Glu?Pro?Glu?Glu?Phe?Leu?Arg?Pro?Arg?Ser?Leu?Gln?Cys?Val?Ser?Leu

165?????????????????170?????????????????175His?Leu?Leu?Ser?Asn?Asp?Met?Cys?Ala?Arg?Ala?Tyr?Ser?Glu?Lys?Val

180?????????????????185?????????????????190Thr?Glu?Phe?Met?Leu?Cys?Ala?Gly?Leu?Trp?Thr?Gly?Gly?Lys?Asp?Thr

195?????????????????200?????????????????205Cys?Gly?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?Asn?Gly?Val?Leu?Gln

210?????????????????215?????????????????220Gly?Ile?Thr?Ser?Trp?Gly?Pro?Glu?Pro?Cys?Ala?Leu?Pro?Glu?Lys?Pro225?????????????????230?????????????????235?????????????????240Ala?Val?Tyr?Thr?Lys?Val?Val?His?Tyr?Arg?Lys?Trp?Ile?Lys?Asp?Thr

245?????????????????250?????????????????255Ile?Ala?Ala?Asn?Pro

260 (2) SEQ ID NO:4 information: (ⅰ) sequence signature:

(A) length: 832 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:4:GGATCCAGC ATG TGG GAC CTG GTT CTC TCC ATC GCC TTG TCT GTG GGG 48

Met?Trp?Asp?Leu?Val?Leu?Ser?Ile?Ala?Leu?Ser?Val?Gly

1???????????????5??????????????????10TGC?ACT?GGT?GCC?GTG?CCC?CTC?ATC?CAG?TCT?CGG?ATT?GTG?GGA?GGC?TGG?????96Cys?Thr?Gly?Ala?Val?Pro?Leu?Ile?Gln?Ser?Arg?Ile?Val?Gly?Gly?Trp

15??????????????????20??????????????????25GAG?TGT?GAG?AAG?CAT?TCC?CAA?CCC?TGG?CAG?GTG?GCT?GTG?TAC?AGT?CAT????144Glu?Cys?Glu?Lys?His?Ser?Gln?Pro?Trp?Gln?Val?Ala?Val?Tyr?Ser?His?30??????????????????35??????????????????40??????????????????45GGA?TGG?GCA?CAC?TGT?GGG?GGT?GTC?CTG?GTG?CAC?CCC?CAG?TGG?GTG?CTC????192Gly?Trp?Ala?His?Cys?Gly?Gly?Val?Leu?Val?His?Pro?Gln?Trp?Val?Leu

50??????????????????55??????????????????60ACA?GCT?GCC?CAT?TGC?CTA?AAG?AAG?AAT?AGC?CAG?GTC?TGG?CTG?GGT?CGG????240Thr?Ala?Ala?His?Cys?Leu?Lys?Lys?Asn?Ser?Gln?Val?Trp?Leu?Gly?Arg

65??????????????????70??????????????????75CAC?AAC?CTG?TTT?GAG?CCT?GAA?GAC?ACA?GGC?CAG?AGG?GTC?CCT?GTC?AGC????288His?Asn?Leu?Phe?Glu?Pro?Glu?Asp?Thr?Gly?Gln?Arg?Val?Pro?Val?Ser

80??????????????????85??????????????????90CAC?AGC?TTC?CCA?CAC?CCG?CTC?TAC?AAT?ATG?AGC?CTT?CTG?AAG?CAT?CAA????336His?Ser?Phe?Pro?His?Pro?Leu?Tyr?Asn?Met?Ser?Leu?Leu?Lys?His?Gln

95?????????????????100?????????????????105AGC?CTT?AGA?CCA?GAT?GAA?GAC?TCC?AGC?CAT?GAC?CTC?ATG?CTG?CTC?CGC????384Ser?Leu?Arg?Pro?Asp?Glu?Asp?Ser?Ser?His?Asp?Leu?Met?Leu?Leu?Arg110?????????????????115?????????????????120?????????????????125CTG?TCA?GAG?CCT?GCC?AAG?ATC?ACA?GAT?GTT?GTG?AAG?GTC?CTG?GGC?CTG????432Leu?Ser?Glu?Pro?Ala?Lys?Ile?Thr?Asp?Val?Val?Lys?Val?Leu?Gly?Leu

130?????????????????135?????????????????140CCC?ACC?CAG?GAG?CCA?GCA?CTG?GGG?ACC?ACC?TGC?TAC?GCC?TCA?GGC?TGG????480Pro?Thr?Gln?Glu?Pro?Ala?Leu?Gly?Thr?Thr?Cys?Tyr?Ala?Ser?Gly?Trp

145?????????????????150?????????????????155GGC?AGC?ATC?GAA?CCA?GAG?GAG?TTC?TTG?CGC?CCC?AGG?AGT?CTT?CAG?TGT????528Gly?Ser?Ile?Glu?Pro?Glu?Glu?Phe?Leu?Arg?Pro?Arg?Ser?Leu?Gln?Cys

160?????????????????165?????????????????170GTG?AGC?CTC?CAT?CTC?CTG?TCC?AAT?GAC?ATG?TGT?GCT?AGA?GCT?TAC?TCT????576Val?Ser?Leu?His?Leu?Leu?Ser?Asn?Asp?Met?Cys?Ala?Arg?Ala?Tyr?Ser

175?????????????????180?????????????????185GAG?AAG?GTG?ACA?GAG?TTC?ATG?TTG?TGT?GCT?GGG?CTC?TGG?ACA?GGT?GGT????624Glu?Lys?Val?Thr?Glu?Phe?Met?Leu?Cys?Ala?Gly?Leu?Trp?Thr?Gly?Gly190?????????????????195?????????????????200?????????????????205AAA?GAC?ACT?TGT?GGG?GGT?GAT?TCT?GGGGGT?CCA?CTT?GTC?TGT?AAT?GGT?????672Lys?Asp?Thr?Cys?Gly?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?Asn?Gly

210?????????????????215?????????????????220GTG?CTT?CAA?GGT?ATC?ACA?TCA?TGG?GGC?CCT?GAG?CCA?TGT?GCC?CTG?CCT????720Val?Leu?Gln?Gly?Ile?Thr?Ser?Trp?Gly?Pro?Glu?Pro?Cys?Ala?Leu?Pro

225?????????????????230?????????????????235GAA?AAG?CCT?GCT?GTG?TAC?ACC?AAG?GTG?GTG?CAT?TAC?CGG?AAG?TGG?ATC????768Glu?Lys?Pro?Ala?Val?Tyr?Thr?Lys?Val?Val?His?Tyr?Arg?Lys?Trp?Ile

240?????????????????245?????????????????250AAG?GAC?ACC?ATC?GCA?GCC?AAC?CCC?TGAGTGCCCC?TGTCCCACCC?CTACCTCTAG???822Lys?Asp?Thr?Ile?Ala?Ala?Asn?Pro

255 260TAAACTGCAG, 832 (2) SEQ ID NO:5 information: (ⅰ) sequence signature:

(A) length: 244 amino acid

(B) type: amino acid

(D) topological framework: linear (ⅱ) molecule type: protein (ⅹ ⅰ) sequence description: SEQ ID NO:5:Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gly Trp Glu Cys Glu Lys 15 10 15His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp Ala His

20??????????????????25??????????????????30Cys?Gly?Gly?Val?Leu?Val?His?Pro?Gln?Trp?Val?Leu?Thr?Ala?Ala?His

35??????????????????40??????????????????45Cys?Leu?Lys?Lys?ASh?Ser?Gln?Val?Trp?Leu?Gly?Arg?His?Asn?Leu?Phe

50??????????????????55??????????????????60Glu?Pro?Glu?Asp?Thr?Gly?Gln?Arg?Val?Pro?Val?Ser?His?Ser?Phe?Pro?65??????????????????70??????????????????75??????????????????80His?Pro?Leu?Tyr?Asn?Met?Ser?Leu?Leu?Lys?His?Gln?Ser?Leu?Arg?Pro

85??????????????????90??????????????????95Asp?Glu?Asp?Ser?Ser?His?Asp?Leu?Met?Leu?Leu?Arg?Leu?Ser?Glu?Pro

100?????????????????105?????????????????110Ala?Lys?Ile?Thr?Asp?Val?Val?Lys?Val?Leu?Gly?Leu?Pro?Thr?Gln?Glu

115?????????????????120?????????????????125Pro?Ala?Leu?Gly?Thr?Thr?Cys?Tyr?Ala?Ser?Gly?Trp?Gly?Ser?Ile?Glu

130?????????????????135?????????????????140Pro?Glu?Glu?Phe?Leu?Arg?Pro?Arg?Ser?Leu?Gln?Cys?Val?Ser?Leu?His145?????????????????150?????????????????155?????????????????160Leu?Leu?Ser?Asn?Asp?Met?Cys?Ala?Arg?Ala?Tyr?Ser?Glu?Lys?Val?Thr

165?????????????????170?????????????????175Glu?Phe?Met?Leu?Cys?Ala?Gly?Leu?Trp?Thr?Gly?Gly?Lys?Asp?Thr?Cys

180?????????????????185?????????????????190Gly?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?Asn?Gly?Val?Leu?Gln?Gly

195?????????????????200?????????????????205Ile?Thr?Ser?Trp?Gly?Pro?Glu?Pro?Cys?Ala?Leu?Pro?Glu?Lys?Pro?Ala

210 215 220Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile225,230 235 240Ala Ala Asn Pro (2) SEQ ID NO:6 information: (ⅰ) sequence signature:

(A) length: 766 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅸ) feature:

(A) title/keyword: CDS

(B) position: 1..732, (ⅹ ⅰ) sequence description: SEQ ID NO:6:GTG CCC CTC ATC CAG TCT CGG ATT GTG GGA GGC TGG GAG TGT GAG AAG 48Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gly Trp Glu Cys Glu Lys 15 10 15CAT TCC CAA CCC TGG CAG GTG GCT GTG TAC AGT CAT GGA TGG GCA CAC 96His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp Ala His

20??????????????????25??????????????????30TGT?GGG?GGT?GTC?CTG?GTG?CAC?CCC?CAG?TGG?GTG?CTC?ACA?GCT?GCC?CAT????144Cys?Gly?Gly?Val?Leu?Val?His?Pro?Gln?Trp?Val?Leu?Thr?Ala?Ala?His

35??????????????????40?????????????????45TGC?CTA?AAG?AAG?AAT?AGC?CAG?GTC?TGG?CTG?GGT?CGG?CAC?AAC?CTG?TTT????192Cys?Leu?Lys?Lys?Asn?Ser?Gln?Val?Trp?Leu?Gly?Arg?His?Asn?Leu?Phe

50??????????????????55??????????????????60GAG?CCT?GAA?GAC?ACA?GGC?CAG?AGG?GTC?CCT?GTC?AGC?CAC?AGC?TTC?CCA????240Glu?Pro?Glu?Asp?Thr?Gly?Gln?Arg?Val?Pro?Val?Ser?His?Ser?Phe?Pro?65??????????????????70??????????????????75??????????????????80CAC?CCG?CTC?TAC?AAT?ATG?AGC?CTT?CTG?AAG?CAT?CAA?AGC?CTT?AGA?CCA????288His?Pro?Leu?Tyr?Asn?Met?Ser?Leu?Leu?Lys?His?Gln?Ser?Leu?Arg?Pro

85??????????????????90??????????????????95GAT?GAA?GAC?TCC?AGC?CAT?GAC?CTC?ATG?CTG?CTC?CGC?CTG?TCA?GAG?CCT????336Asp?Glu?Asp?Ser?Ser?His?Asp?Leu?Met?Leu?Leu?Arg?Leu?Ser?Glu?Pro

100?????????????????105?????????????????110GCC?AAG?ATC?ACA?GAT?GTT?GTG?AAG?GTC?CTG?GGC?CTG?CCC?ACC?CAG?GAG????384Ala?Lys?Ile?Thr?Asp?Val?Val?Lys?Val?Leu?Gly?Leu?Pro?Thr?Gln?Glu

115?????????????????120?????????????????125CCA?GCA?CTG?GGG?ACC?ACC?TGC?TAC?GCC?TCA?GGC?TGG?GGC?AGC?ATC?GAA????432Pro?Ala?Leu?Gly?Thr?Thr?Cys?Tyr?Ala?Ser?Gly?Trp?Gly?Ser?Ile?Glu

130?????????????????135?????????????????140CCA?GAG?GAG?TTC?TTG?CGC?CCC?AGG?AGT?CTT?CAG?TGT?GTG?AGC?CTC?CAT????480Pro?Glu?Glu?Phe?Leu?Arg?Pro?Arg?Ser?Leu?Gln?Cys?Val?Ser?Leu?His145?????????????????150?????????????????155?????????????????160CTC?CTG?TCC?AAT?GAC?ATG?TGT?GCT?AGA?GCT?TAC?TCT?GAG?AAG?GTG?ACA????528Leu?Leu?Ser?Asn?Asp?Met?Cys?Ala?Arg?Ala?Tyr?Ser?Glu?Lys?Val?Thr

165?????????????????170?????????????????175GAG?TTC?ATG?TTG?TGT?GCT?GGG?CTC?TGG?ACA?GGT?GGT?AAA?GAC?ACT?TGT????576Glu?Phe?Met?Leu?Cys?Ala?Gly?Leu?Trp?Thr?Gly?Gly?Lys?Asp?Thr?Cys

180?????????????????185?????????????????190GGG?GGT?GAT?TCT?GGG?GGT?CCA?CTT?GTC?TGT?AAT?GGT?GTG?CTT?CAA?GGT????624Gly?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Val?Cys?AsT?Gly?Val?Leu?Gln?Gly

195?????????????????200?????????????????205ATC?ACA?TCA?TGG?GGC?CCT?GAG?CCA?TGT?GCC?CTG?CCT?GAA?AAG?CCT?GCT????672Ile?Thr?Ser?Trp?Gly?Pro?Glu?Pro?Cys?Ala?Leu?Pro?Glu?Lys?Pro?Ala

210 215 220GTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TGG ATC AAG GAC ACC ATC 720Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile225,230 235 240GCA GCC AAC CCC TGAGTGCCCC TGTCCCACCC CTACCTCTAG TAAA 766Ala Ala Asn Pro (2) SEQ ID NO:7 information: (ⅰ) sequence signature:

(A) length: 237 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: peptide

(ⅹ ⅰ) sequence description: SEQ ID NO:7:Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val 15 10 15Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly Val Leu Val His

20??????????????????25??????????????????30Pro?Gln?Trp?Val?Leu?Thr?Ala?Ala?His?Cys?Ile?Arg?Asn?Lys?Ser?Val

35??????????????????40??????????????????45Ile?Leu?Leu?Gly?Arg?His?Ser?Leu?Phe?His?Pro?Glu?Asp?Thr?Gly?Gln

50??????????????????55??????????????????60Val?Phe?Gln?Val?Ser?His?Ser?Phe?Pro?His?Pro?Leu?Tyr?Asp?Met?Ser65??????????????????70??????????????????75??????????????????80Leu?Leu?Lys?Asn?Arg?Phe?Leu?Arg?Pro?Gly?Asp?Asp?Ser?Ser?His?Asp

85??????????????????90??????????????????95Leu?Met?Leu?Leu?Arg?Leu?Ser?Glu?Pro?Ala?Glu?Leu?Thr?Asp?Ala?Val

100?????????????????105?????????????????110Lys?Val?Met?Asp?Leu?Pro?Thr?Gln?Glu?Pro?Ala?Leu?Gly?Thr?Thr?Cys

115?????????????????120?????????????????125Tyr?Ala?Ser?Gly?Trp?Gly?Ser?Ile?Glu?Pro?Glu?Glu?Phe?Leu?Thr?Pro

130?????????????????135?????????????????140Lys?Lys?Leu?Gln?Cys?Val?Asp?Leu?His?Val?Ile?Ser?Asn?Asp?Val?Cys145?????????????????150?????????????????155?????????????????160Ala?Gln?Val?His?Pro?Gln?Lys?Val?Thr?Lys?Phe?Met?Leu?Cys?Ala?Gly

165?????????????????170?????????????????175Arg?Trp?Thr?Gly?Gly?Lys?Ser?Thr?Cys?Ser?Gly?Asp?Ser?Gly?Gly?Pro

180?????????????????185?????????????????190Leu?Val?Cys?Asn?Gly?Val?Leu?Gln?Gly?Ile?Thr?Ser?Trp?Gly?Ser?Glu

195?????????????????200?????????????????205Pro?Cys?Ala?Leu?Pro?Glu?Arg?Pro?Ser?Leu?Tyr?Thr?Lys?Val?Val?His

210 215 220Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn Pro225,230 235 (2) SEQ ID NO:8 information: (ⅰ) sequence signature:

(A) length: 237 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:8:Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val1 5 10 15Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His

195?????????????????200?????????????????205Pro?Cys?Ala?Leu?Pro?Glu?Lys?Pro?Val?Val?Tyr?Thr?Lys?Val?Val?His

210 215 220Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro225,230 235 (2) SEQ ID NO:9 information: (ⅰ) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:9:ACGCGGATCC AGCATGTGGG ACCTGGTTCT CT 32 (2) SEQ ID NO:10 information: (ⅰ) sequence signature:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:10:ACAGCTGCAG TTTACTAGAG GTAGGGGTGG GAC 33 (2) SEQ ID NO:11 information: (ⅰ) sequence signature:

(A) length: 42 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:11:ATATGGATCC ATATGTCAGC ATGTGGGACC TGGTTCTCTC CA 42 (2) SEQ ID NO:12 information: (ⅰ) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:12:ATATGGATCC TCAGGGGTTG GCTGCGATGG T 31 (2) SEQ ID NO:13 information: (ⅰ) sequence signature:

(A) length: 14 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:13:TCATCTCTGT ATCC 14 (2) SEQ ID NO:14 information: (ⅰ) sequence signature:

(A) length: 12 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:14:GAGTAAGCTC TA 12 (2) SEQ ID NO:15 information: (ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:15:GATGACTCCA GCCACGACCT 20 (2) SEQ ID NO:16 information: (ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:16:CACAGACACC CCATCCTATC 20 (2) SEQ ID NO:17 information: (ⅰ) sequence signature:

(A) length: 23 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:17:GAGGGTTGTG TACAGTCATG GAT 23 (2) SEQ ID NO:18 information: (ⅰ) sequence signature:

(A) length: 23 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: cDNA (ⅹ ⅰ) sequence description: SEQ ID NO:18:ACACACTGAA GACTCCTGGG GCG 23 (2) SEQ ID NO:19 information: (ⅰ) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅴ) clip types: inner (ⅹ ⅰ) sequence description: SEQ ID NO:19:Glu Lys His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp 15 10 15Ala His Cys (2) SEQ ID NO:20 information: (ⅰ) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅴ) clip types: inner (ⅹ ⅰ) sequence description: SEQ ID NO:20:His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu 15 10 15 (2) SEQ ID NO:21 information: (ⅰ) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅴ) clip types: inner (ⅹ ⅰ) sequence description: SEQ ID NO:21:His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys 15 10 15 (2) SEQ ID NO:22 information: (ⅰ) sequence signature:

(A) length: 55 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅴ) clip types: inner (ⅹ ⅰ) sequence description: SEQ ID NO:22:Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His 15 10 15Pro Gln Trp Val Leu Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gln

20??????????????????25??????????????????30Val?Trp?Leu?Gly?Arg?His?Asn?Leu?Phe?Glu?Pro?Glu?Asp?Thr?Gly?Gln

35??????????????????40??????????????????45Arg?Val?Pro?Val?Ser?His?Ser

50 55 (2) SEQ ID NO:23 information: (ⅰ) sequence signature:

(A) length: 711 base pairs

(B) type: amino acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO:23:ATTGTGGGAG GCTGGGAGTG CGAGAAGCAT TCCCAACCCT GGCAGGTGCT TGTGGCCTCT 60CGTGGCAGGG CAGTCTGCGG CGGTGTTCTG GTGCACCCCC AGTGGGTCCT CACAGCTGCC 120CACTGCATCA GGAACAAAAG CGTGATCTTG CTGGGTCGGC ACAGCCTGTT TCATCCTGAA 180GACACAGGCC AGGTATTTCA GGTCAGCCAC AGCTTCCCAC ACCCGCTCTA CGATATGAGC 240CTCCTGAAGA ATCGATTCCT CAGGCCAGGT GATGACTCCA GCCACGACCT CATGCTGCTC 300CGCCTGTCAG AGCCTGCCGA GCTCACGGAT GCTGTGAAGG TCATGGACCT GCCCACCCAG 360GAGCCAGCAC TGGGGACCAC CTGCTACGCC TCAGGCTGGG GCAGCATTGA ACCAGAGGAG 420TTCTTGACCC CAAAGAAACT TCAGTGTGTG GACCTCCATG TTATTTCCAA TGACGTGTGT 480GCGCAAGTTC ACCCTCAGAA GGTGACCAAG TTCATGCTGT GTGCTGGACG CTGGACAGGG 540GGCAAAAGCA CCTGCTCGGG TGATTCTGGG GGCCCACTTG TCTGTAATGG TGTGCTTCAA 600GGTATCACGT CATGGGGCAG TGAACCATGT GCCCTGCCCG AAAGGCCTTC CCTGTACACC 660AAGGTGGTGC ATTACCGGAA GTGGATCAAG GACACCATCG TGGCCAACCC C 711 ( 2 ) SEQ ID NO:24： ( ⅰ ) ：

(A) length: 24 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅴ) clip types: inner (ⅹ ⅰ) sequence description: SEQ ID NO:24:Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe Glu 15 10 15Pro Glu Asp Thr Gly Gln Arg Val

20 (2) SEQ ID NO:25 information: (ⅰ) sequence signature:

(A) length: 26 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅴ) clip types: inner (ⅹ ⅰ) sequence description: SEQ ID NO:25:Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His Tyr 15 10 15Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala

20 25 (2) SEQ ID NO:26 information: (ⅰ) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: peptide (ⅴ) clip types: inner (ⅹ ⅰ) sequence description: SEQ ID NO:26:Gln Val Ala Val Tyr Ser His Gly Trp Ala His Cys 15 10

Claims

1. A diagnostic method for detecting hK2 DNA comprising:

(a) DNA is generated by reverse transcription from RNA derived from human physiological samples containing cells suspected of containing hK2 RNA, and a certain amount of the DNA is combined with a certain amount under conditions that can effectively amplify the DNA by polymerase chain reaction Amounts of at least two oligonucleotides are contacted to obtain an amount of amplified hK2 DNA, wherein at least one oligonucleotide is a hK2-specific oligonucleotide; and

(b) Detecting the presence of amplified hK2 DNA.

2. A method of detecting metastatic prostate cancer in a human, comprising:

(a) DNA is generated by reverse transcription from RNA derived from human physiological samples containing cells suspected of containing hK2 RNA, and a certain amount of the DNA is mixed with a certain amount under conditions that can effectively amplify the DNA by polymerase chain reaction at least two oligonucleotides of which are hK2-specific oligonucleotides, thereby obtaining an amount of amplified hK2 DNA; and

(b) detecting the presence of amplified hK2 DNA, wherein the presence of hK2 DNA is indicative of metastatic prostate cancer in said human.

3. The method of claim 1 or 2, wherein the physiological sample is a tissue sample.

4. 3. The method of claim 3, wherein said tissue is selected from the group consisting of prostate, prostate capsule, seminal vesicles, bone marrow, and lymph nodes.

5. 3. The method of claim 3, wherein said tissue is non-prostatic tissue.

6. The method of claim 1 or 2, wherein said physiological sample is a liquid.

7. 6. The method of claim 6, wherein said fluid is selected from the group consisting of whole blood, serum and semen.

8. The method of claim 7, wherein said fluid is whole blood.

9. The method of claim 1 or 2, wherein the amplified hK2 DNA is subjected to agarose gel electrophoresis before detection.

10. The method of claim 1 or 2, further comprising determining the amount of amplified hK2 DNA.

11. The method of claim 1 or 2, further comprising:

(c) under the conditions that can effectively amplify prostate-specific antigen (PSA) DNA by polymerase chain reaction and amplify hK2 DNA, another quantitative and a certain amount of DNA obtained by reverse transcription of RNA derived from human physiological samples At least two of the oligonucleotides are contacted to generate amplified PSADNA; and

(d) Detecting the presence of amplified PSA DNA.

12. A diagnostic method for detecting hK2 RNA comprising:

(a) RNA is extracted from a physiological sample of human origin;

(b) performing reverse transcription on the extracted RNA to generate DNA;

(c) contacting the DNA with an amount of at least two oligonucleotides to generate an amount of amplified hK2 DNA under conditions effective to amplify the DNA by polymerase chain reaction, wherein at least one of the oligonucleotides the nucleotides are hK2-specific oligonucleotides; and

(d) detecting the presence of amplified hK2 DNA, wherein the presence of amplified hK2 DNA is indicative of metastatic prostate cancer in the human.

13. The method of claim 12, wherein said sample is a tissue sample.

14. The method of claim 12, wherein said sample is a physiological fluid sample.

15. 12. The method of claim 12, wherein said person has undergone a total prostatectomy and the presence of hK2 DNA indicates that the person has persistent prostate cancer.

16. A method of monitoring the development of prostate cancer comprising:

(a) reverse-transcribing RNA derived from a physiological sample of a prostate cancer patient to generate DNA, and combining a certain amount of the DNA with a certain amount of at least two oligonucleotides under conditions that can effectively amplify the DNA by polymerase chain reaction; Nucleotide contacting to produce an amount of amplified hK2 DNA wherein at least one oligonucleotide is an hK2-specific oligonucleotide;

(b) detecting or measuring the amount of amplified hK2 DNA;

(c) repeating steps (a) and (b) after a certain period of time; and

(d) comparing the results of steps (b) and (c), wherein an increase in the amount of hK2 DNA indicates that prostate cancer is developing in said human.

17. A method for pathologically determining the stage of prostate cancer comprising:

(a) DNA is generated by reverse transcription from RNA derived from a physiological sample of a prostate cancer patient, and a certain amount of the DNA is mixed with a certain amount of at least two oligonucleotides under conditions that can effectively amplify the DNA by polymerase chain reaction. Nucleotide contacting to generate an amount of amplified hK2 DNA wherein at least one oligonucleotide is an hK2-specific oligonucleotide; and

(b) detecting the presence or quantifying the amount of amplified hK2 DNA, wherein the presence or amount of amplified hK2 DNA is indicative of the pathological stage of the prostate cancer.

18. The method of claim 16 or 17, wherein said human is a candidate for total prostatectomy.

19. 16. The method of claim 16, wherein the sample of step (a) is obtained before the patient receives hormone therapy.

20. The method of claim 19, wherein the hormone therapy is androgen therapy.

twenty one. The method of claim 20, wherein the androgen therapy is androgen stimulating therapy.

twenty two. The method of claim 16 or 17, wherein said sample is a non-prostatic tissue sample.

twenty three. The method of claim 16 or 17, wherein said sample is a physiological fluid sample.

twenty four. 23. The method of claim 23, wherein said fluid is whole blood.

25． A diagnostic kit for detecting hK2 RNA in a physiological sample suspected of having hK2 RNA, comprising (a) a known amount of a first hK2-specific oligonucleotide comprising at least about 7-50 nuclei and the oligonucleotide is at least about 80% identical to SEQ ID NO:4, and (b) a known amount of a second hK2-specific oligonucleotide containing at least about 7 -50 nucleotides, and the oligonucleotide is at least about 80% identical to a nucleotide sequence complementary to SEQ ID NO:4.

26． The diagnostic kit of claim 25, wherein the second oligonucleotide comprises SEQ ID NO:14.

27． The diagnostic kit of claim 25, wherein the first oligonucleotide comprises SEQ ID NO: 17.

28． The diagnostic kit of claim 25, wherein the second oligonucleotide comprises SEQ ID NO: 18.

29． An isolated, purified peptide comprising SEQ ID NO: 22, a biologically active subunit thereof, or a biologically active variant thereof.

30． An isolated, purified peptide comprising SEQ ID NO: 26, a biologically active subunit thereof, or a biologically active variant thereof.

31． A purified antibody specifically reactive with the protein or polypeptide comprising the peptide of claim 29 or 30.

32． The antibody of claim 31 which is a monoclonal antibody.

33． A hybridoma cell line producing the antibody of claim 32.

34． A polyclonal antibody preparation comprising the antibody of claim 31.

35． An oligonucleotide comprising at least about 7-50 nucleotides and at least about 80% identical to or complementary to a nucleotide sequence having SEQ ID NO:4.

36． The oligonucleotide of claim 35 comprising SEQ ID NO:14.

37． The oligonucleotide of claim 35 comprising SEQ ID NO: 17.

38． The oligonucleotide of claim 35 comprising SEQ ID NO: 18.

39． A method of detecting or determining the presence of metastatic prostate cancer in a human non-prostate tissue sample comprising:

(a) mixing an amount of an agent that binds hK2 polypeptide but not hK3 with cells of a human tissue sample to form a bivalent complex comprising the agent and cells.

(b) detecting or determining the presence or amount of the complex in a sample, wherein the presence or amount of the complex is indicative of the presence of micrometastatic prostate cancer.

40． 39. The method of claim 39, wherein said reagent is an antibody.

41． 39. The method of claim 39, wherein said antibody is a member of a population of polyclonal antibodies.

42． 39. The method of claim 39, wherein said antibody is a monoclonal antibody.